Ethanol production using xylitol synthesis mutant of xylose-utilizing zymomonas

ABSTRACT

Production of ethanol using a strain of xylose-utilizing  Zymomonas  with a genetic modification of the glucose-fructose oxidoreductase gene was found to be improved due to greatly reduced production of xylitol, a detrimental by-product of xylose metabolism synthesized during fermentation.

This application claims the benefit of U.S. Provisional Application No.60/847,856, filed Sep. 28, 2006, which is incorporated in its entiretyas a part hereof for all purposes.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support underContract Nos. 04-03-CA-70224 and DE-FC36-03GO13146 awarded by theDepartment of Energy. The government has certain rights in thisinvention.

FIELD OF INVENTION

The invention relates to the fields of microbiology and geneticengineering. More specifically, production of ethanol using a strain ofxylose-utilizing Zymomonas with a genetic modification of theglucose-fructose oxidoreductase gene was found to be improved due togreatly reduced production of xylitol, a detrimental by-product ofxylose metabolism synthesized during fermentation.

BACKGROUND OF INVENTION

Production of ethanol by microorganisms provides an alternative energysource to fossil fuels and is therefore an important area of currentresearch. Zymomonas mobilis is a bacterial ethanologen that grows onglucose, fructose, and sucrose, metabolizing these sugars to CO₂ andethanol via the Entner-Douderoff pathway. Though wild type strainscannot use xylose as a carbon source, recombinant strains of Z. mobilisthat are able to grow on this sugar have been engineered (U.S. Pat. No.5,514,583, U.S. Pat. No. 5,712,133, WO 95/28476, Feldmann et al. (1992)Appl Microbiol Biotechnol 38: 354-361, Zhang et al. (1995) Science267:240-243). Xylose is the major pentose in hydrolyzed lignocellulosicmaterials, and therefore can provide an abundantly available, low costcarbon substrate for use in fermentation. Z. mobilis has been engineeredfor expression of four enzymes needed for xylose metabolism: 1) xyloseisomerase, which catalyses the conversion of xylose to xylulose; 2)xylulokinase, which phosphorylates xylulose to form xylulose5-phosphate; 3) transketolase; and 4) transaldolase (U.S. Pat. No.5,514,583, U.S. Pat. No. 6,566,107; Zhang et al. (1995) Science267:240-243). Through the combined actions of these four enzymes and thecell's normal metabolic machinery, three molecules of xylose areconverted to two molecules of glucose 6-phosphate and one molecule ofglyceraldehyde 3-phosphate, which are subsequently converted to ethanoland CO₂ on the glucose side of the pathway (see FIG. 1).

Though there has been success in engineering Z. mobilis strains forxylose metabolism, the strains do not grow and produce ethanol as wellon xylose as on glucose. One factor that causes poor growth on xylose isthe production of xylitol as a by-product of xylose metabolism (Feldmannet al. supra; Kim et al. (2000) Applied and Environmental Microbiology66:186-193). Xylitol is phosphorylated by xylulose kinase to producexylitol 5-phosphate, which accumulates in the cell and inhibitsbacterial growth. Xylitol synthesis also reduces the yield of ethanol,since xylose-utilizing recombinant strains of Z. mobilis cannot convertxylitol to ethanol. In addition, xylitol is a potent inhibitor of xyloseisomerase (Smith et al. (1991) Biochem J. 277:255-261), which catalyzesthe first step of xylose utilization in the engineered xylose metabolismpathway. See FIG. 2 for a diagram showing xylitol synthesis and effects.

The physiological pathway and enzymes that are responsible for xylitolsynthesis in vivo have not been determined. However, it has beendemonstrated that cell-free extracts from wild type Z. mobilis are ableto reduce xylose to xylitol when they are supplemented with NADPH(Feldmann et al., supra), and that this reaction is catalyzed by anNADPH-dependent aldose reductase. It has also been shown that Z. mobiliscell-free extracts are able to convert a small amount of xylose toxylitol without NADPH supplementation, and that xylitol production underthese conditions increases 3- to 4-fold when purified xylose isomeraseis also added to the reaction mixture (Danielson, 2001, University ofColorado Masters Thesis). Since xylose isomerase is able to convertxylose to xylulose, the clear implication of the latter experiment isthat the Z. mobilis enzyme glucose-fructose oxidoreductase (GFOR) canuse xylose as an electron donor and xylulose as an electron acceptor togenerate xylitol as will be discussed in greater detail below. Thus,there are at least two pathways for xylitol production in Z. mobilisbased on the in vitro experiments, but the extent to which theycontribute to xylitol formation under physiological conditions remainsto be determined.

For high-level production of ethanol, Z. mobilis is grown in highconcentrations of a fermentable carbon source, which can result inosmotic shock. Osmotic shock manifests itself as a long lag periodbefore growth commences when wild type strains are transferred to liquidmedia that contains >200 g/L of glucose or fructose or >360 g/L ofsucrose (Loos et al. (1994) J Bacteriol 176:7688-7693). Furthermore,addition of sorbitol to the growth medium reduces the lag period whenwild type strains are shifted to high concentrations of these sugars(Wiegert et al. (1996) Arch Microbiol 166:32-41, Loos et al supra).

It has also been shown that the periplasmic enzyme glucose-fructoseoxidoreductase (GFOR) plays an important role in osmotic balance whenwild type Z. mobilis is grown in concentrated mixtures of glucose andfructose (Loos et al. supra) or concentrated solutions of sucrose(Wiegert et al. supra, Loos et al. supra). Briefly, GFOR with itstightly bound co-factor, catalyzes the oxidation of glucose togluconolactone and subsequent reduction of fructose to sorbitol in aclassical Ping Pong Bi mechanism as shown in Diagram I. The sorbitolthat is generated in the periplasmic space is transported into cellsagainst a concentration gradient where it accumulates to high levelssince it is not further metabolized. The high concentration of sorbitolinside the cells eliminates the osmotic pressure difference across theplasma membrane and restores osmotic balance.

A spontaneous mutant of wild type Z. mobilis that cannot generatesorbitol was show to produce higher levels of ethanol than wild typecells when it was grown on low concentrations of sucrose (<150 g/L), butthis strain could not grow on high concentrations of sucrose (Kirk andDoelle (1993) Biotechnol. Letters 15:985-990). This mutant wassubsequently shown to lack expression of glucose-fructose oxidoreductase(GFOR), which accounts for its inability to convert any of thesucrose-derived fructose to the unwanted by-product sorbitol (Wiegert etal. supra). It was also shown that growth of the sorbitol-deficientmutant in high concentrations of sucrose could be restored by addingsorbitol to the growth medium (Wiegert et al., supra). Thus GFOR plays acritical role in osmotic balance by synthesizing sorbitol when Z.mobilis is grown in concentrated mixtures of glucose and fructose orhigh concentrations of sucrose, which is hydrolyzed to glucose andfructose by the host cell's invertase.

CN1600850(A) discloses a non-xylose utilizing mutant strain of Z.mobilis that has an inactivated GFOR gene, and production of ethanolusing this strain. The lack of sorbitol production with this strainresulted in higher levels of ethanol when glucose, fructose or sucrosewas the carbon source.

The effects of reducing or eliminating glucose-fructose oxidoreductaseenzyme activity in an engineered xylose-utilizing strain of Z. mobilisthat is grown on a mixture of xylose and glucose (in the absence of anyadded sucrose or fructose) are not known.

The problem to be addressed is how to produce increased amounts ofethanol in fermentations using a xylose-utilizing Z. mobilis straingrown on xylose-containing medium. Applicants have solved the problem bydetermining the principle pathway for production of xylitol in vivo, andeliminating the enzyme activity that is responsible for its formationthrough gene inactivation, thereby creating a Z. mobilis strain that maybe used for improved ethanol production.

SUMMARY OF INVENTION

The present invention relates to a process for production of ethanolusing a strain of Zymomonas that has reduced production of xylitol andincreased production of ethanol when grown in the presence of xylose.Applicants have discovered that xylitol production in xylosemetabolizing Z. mobilis is predominantly mediated by the enzymeglucose-fructose oxidoreductase (GFOR). A genetically modified strainthat does not express GFOR (a GFOR knockout mutant) was constructed andfound to produce reduced amounts of xylitol when grown onxylose-containing sugar mixtures. The GFOR knockout mutant also consumedmore xylose and produced higher concentrations of ethanol when grown inhigh sugar mixtures in the presence of sorbitol than the parent strainthat expresses GFOR. In addition, the ethanol yield (the amount ofethanol produced per gram of sugar consumed) was significantly higherfor the GFOR knockout strain.

Accordingly the invention provides a process for producing ethanolcomprising,

a) providing a recombinant Zymomonas strain capable of utilizing xyloseto produce ethanol, said strain comprising at least one geneticmodification which reduces glucose-fructose oxidoreductase activity, and

b) culturing the strain of (a) in a medium comprising xylose whereby thexylose is converted to ethanol.

The process above provides improved conversion of xylose to ethanolrelative to a recombinant Zymomonas strain without at least one geneticmodification which reduces glucose-fructose oxidoreductase activity.

The process specifically improves one or more of the parameters rate,titer or yield, of xylose conversion to ethanol relative to arecombinant Zymomonas strain without at least one genetic modificationwhich reduces glucose-fructose oxidoreductase activity.

In an aspect of the invention, the process described herein is improvedin that strain of (a) above produces a reduced amount of xylitol, or noxylitol, as compared to Zymomonas strain without at least one geneticmodification which reduces glucose-fructose oxidoreductase activity.

BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS AND SEQUENCEDESCRIPTIONS

The invention can be more fully understood from the following detaileddescription, the figures, and the accompanying sequence descriptionsthat form a part of this application.

FIG. 1 shows a diagram of the four enzymes (boxed) that have been usedto engineer Z. mobilis for xylose utilization and biochemical pathwaysfor ethanol production using xylose.

FIG. 2 shows a diagram of the first two steps of the engineered xylosepathway (boxed), xylitol synthesis, xylitol 5-phosphate formation (atoxic deadend intermediate), and inhibition of xylose isomerase byxylitol.

FIG. 3 shows the strategies for enzyme assays of transketolase (A),transaldolase (B), xylose isomerase (C), and xyulokinase (D).

FIG. 4 shows a plasmid map of pMODPgaptaltktCm.

FIG. 5 shows a plasmid map of pMODPgapxylABCm.

FIG. 6 shows a graph of xylose isomerase (XI) and xylulokinase (XK)activities in T2C, T3C, T4C, and T5C lines transformed with PgapxylAB.

FIG. 7 shows a graph of transaldolse (TAL) and transketolase (TKT)activities in T2C, T3C, T4C, and T5C lines transformed with PgapxylAB.

FIG. 8 shows a graph of % theoretical ethanol yield and % xyloseutilization of selected adapted xylose-utilizing strain colonies.

FIG. 9 shows a graph of growth of adapted xylose-utilizing strains at 70hr on RM (rich medium) with 5% xylose (RMX5%) before and after growing50 generations in RM with 5% glucose (RMG).

FIG. 10 shows graphs of growth, glucose or xylose utilization, andethanol and xylitol production for the selected strain, ZW658 incomparison to the control, 8b, in RM+10% glucose (RMG10%) (A, B) andRM+8% xylose (RMX8%) (C, D).

FIG. 11 shows graphs of growth, glucose and xylose utilization, andethanol and xylitol production for the selected strain, ZW658 incomparison to the control, 8b, in RM+10% glucose and 8% xlose withoutacetate (A, B) or with 0.6% acetate (C, D).

FIG. 12 shows maps of plasmids made during construction of a suicideconstruct for insertional-inactivation of the GFOR gene, and the finalproduct: GFORSp-9WW.

FIG. 13 shows maps of plasmids made for construction of a xyloseisomerase expression plasmid: pZB188/Kan-XylA, and a diagram of the E.coli xylose isomerase expression cassette that was used for thisconstruct (boxed).

FIG. 14 shows graphs of xylitol and xylulose production by ZW1 strainswith and without GFOR gene inactivation, in the presence and absence ofxylose isomerase expression.

FIG. 15 shows graphs of growth, glucose and xylose utilization, andethanol production of xylose-utilizing Z. mobilis strains without (A)and with (B) GFOR gene inactivation, grown on 97 g/L totalxylose+glucose.

FIG. 16 shows graphs of growth, glucose and xylose utilization, andethanol production of xylose-utilizing Z. mobilis strains without (A)and with (B) GFOR gene inactivation, grown on 188 g/L totalxylose+glucose.

FIG. 17 shows a graph of growth of a xylose-utilizing Z. mobilis strainwith GFOR gene inactivation in the presence of different concentrationsof sorbitol.

FIG. 18 shows graphs of xylose utilization, ethanol production, andxylitol production of xylose-utilizing Z. mobilis strains without (A, C)and with (B, D) GFOR gene inactivation, in the absence (A, B) andpresence (C, D) of acetate in 174 g/L of total xylose+glucose.

FIG. 19 shows graphs of xylose utilization, ethanol production, andxylitol production of xylose-utilizing Z. mobilis strains without (A, C)and with (B, D) GFOR gene inactivation, in the absence (A, B) andpresence (C, D) of acetate in 203 g/L of total xylose+glucose.

FIG. 20 shows graphs of xylose utilization, ethanol production, andxylitol production of xylose-utilizing Z. mobilis strains without (A, C)and with (B, D) GFOR gene inactivation, in the absence (A, B) andpresence (C, D) of acetate in 203 g/L of total xylose+glucose withadditional potassium bicarbarbonate for increased buffering capacity.

FIG. 21 shows a graph of xylose and glucose utilization, ethanolproduction, and xylitol production of a xylose-utilizing Z. mobilisstrain with GFOR gene inactivation, in the presence of acetate in 189g/L of total xylose+glucose in a pH-controlled fermentation run.

FIG. 22 shows plasmid maps of pZB188/Kan and pZB188/kan-Cre, a CreExpression vector that can replicate in Z. mobilis.

FIG. 23A shows a comparison of the growth of ZW801-4 and ZW800 in highglucose+xylose, with acetate under pH-controlled conditions. FIG. 23Bshows a graph of, glucose and xylose utilization, and ethanol productionfor ZW801-4 in comparison to ZW800.

FIG. 24 shows an alignment of the translated mutant sequence in ZW801-4with the wild type GFOR protein.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions which form a partof this application.

The following sequences conform with 37C.F.R. 1.821-1.825 (“Requirementsfor Patent Applications Containing Nucleotide Sequences and/or AminoAcid Sequence Disclosures—the Sequence Rules”) and consistent with WorldIntellectual Property Organization (WIPO) Standard ST.25 (1998) and thesequence listing requirements of the EPO and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

A Sequence Listing is provided herewith on Compact Disk. The contents ofthe Compact Disk containing the Sequence Listing are hereby incorporatedby reference in compliance with 37 CFR 1.52(e). The Compact Discs aresubmitted in duplicate and are identical to one another. The discs arelabeled “Copy 1—Sequence Listing” and “Copy 2 Sequence listing” Thediscs contain the following file: CL3662 seq list.ST25.

SEQ ID NOs:1 and 2 are the nucleotide sequences of primers foramplification of a DNA fragment containing theglyceraldehyde-3-phosphate dehydrogenase gene promoter (P_(gap)) frompZB4.

SEQ ID NOs:3 and 4 are the nucleotide sequences of primers foramplification of a DNA fragment containing a tal coding region frompZB4.

SEQ ID NOs:5 and 6 are the nucleotide sequences of primers foramplification of a DNA fragment containing P_(gap)tal from the P_(gap)and tal fragments.

SEQ ID NOs:7 and 8 are the nucleotide sequences of primers foramplification of a DNA fragment containing loxP::Cm from pZB186.

SEQ ID NO:9 is the complete nucleotide sequence for thepMODP_(gap)taltktCm plasmid.

SEQ ID NOs:10 and 11 are the nucleotide sequences of primers foramplification of a 3 kb DNA fragment containing tal and tkt codingregions in transformants receiving pMODP_(gap)taltktCm.

SEQ ID NO:12 is the complete nucleotide sequence for thepMODP_(gap)xylABCm plasmid.

SEQ ID NOs:13 and 14 are the nucleotide sequences of primers foramplification of a 1.6 kb P_(gap)xylA DNA fragment from the T2C, T3C,T4C and T5C integrants with pMODP_(gap)xylABCm.

SEQ ID NOs:15 and 16 are the nucleotide sequences of primers foramplification of a 1.3 kb xylB DNA fragment from the T2C, T3C, T4C andT5C integrants with pMODP_(gap)xylABCm.

SEQ ID NOs:17 and 18 are the nucleotide sequences of primers foramplification of a 2268 bp DNA frag from Z. mobilis W1 genomic DNAcontaining a portion of the 3′ end of the pgm gene, the Idh gene, and aportion of the 5′ end of the adhI gene.

SEQ ID NOs:19 and 20 are the nucleotide sequences of primers foramplification of the tetracycline resistance cassette from pACYC184.

SEQ ID NOs:21 and 22 are oligonucleotide sequences used to create a loxPsite.

SEQ ID NOs:23 and 24 are oligonucleotide sequences used to create a loxPsite.

SEQ ID NOs:25 and 26 are the nucleotide sequences of primers foramplification of the Spec^(r)-cassette from pHP15578.

SEQ ID NOs:27 and 28 are the nucleotide sequences of primers foramplification of 3′ GFOR flanking DNA from ZW1 genomic DNA.

SEQ ID NOs:29 and 30 are the nucleotide sequences of primers foramplification of 5′ GFOR flanking DNA from ZW1 genomic DNA.

SEQ ID NO:31 is the nucleotide sequence of the pGFORSp-9WW plasmid.

SEQ ID NOs:32 and 33 are the nucleotide sequences of primers foramplification of the Kan^(r)-cassette from pET-24a.

SEQ ID NO:34 is the nucleotide sequence of the E. coli xylA expressioncassette that was derived from pZB4.

SEQ ID NOs:35 and 36 are the nucleotide sequences of primers foramplification of a Cre-expression cassette.

SEQ ID NO:37 is the complete nucleotide sequence of the disrupted GFORcoding region in ZW801-4 (from the original start codon through theoriginal stop codon), SEQ ID NO:38 is the complete nucleotide sequenceof the wild type GFOR coding region (from the original start codonthrough the original stop codon),

Applicants made the following biological deposit under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for the Purposes of Patent Procedure at the AmericanType Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va.20110-2209:

Depositor Identification International Depository Date of ReferenceDesignation Deposit ZW658 ATCC # PTA-7858 Sep. 12, 2006

DETAILED DESCRIPTION

The present invention describes a process for producing ethanol fromfermentation using genetically modified xylose-utilizing Zymomonasstrains with reduced GFOR enzyme activity. The xylose-utilizingrecombinant Zymomonas used in the processes further engineered with atleast one modification in the glucose-fructose oxidoreductase (GFOR)gene such that expression of the active enzyme is reduced or eliminated.Applicants show that elimination of GFOR enzyme activity results inreduced xylitol production during xylose metabolism and enhanced ethanolproduction. Ethanol produced by the new Zymomonas strain may be used asan alternative energy source to fossil fuels.

The following abbreviations and definitions will be used for theinterpretation of the specification and the claims.

“Glucose-fructose oxidoreductase” is abbreviated GFOR.

RM is rich medium.

RMG5% is RM+5% glucose.

RMG10% is RM+10% glucose.

RMX8% is RM+8% xylose.

RMX2% is RM+2% xylose.

RMX5% is RM+5% xylose.

RMGX10%8% is RM+10% glucose and 8% xylose.

RMGX5%8% is RM+5% glucose and 8% xylose.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” or “wild type gene” refers to a gene as found in naturewith its own regulatory sequences. “Chimeric gene” refers to any genethat is not a native gene, comprising regulatory and coding sequencesthat are not found together in nature. Accordingly, a chimeric gene maycomprise regulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes.

The term “genetic construct” refers to a nucleic acid fragment thatencodes for expression of one or more specific proteins. In the geneconstruct the gene may be native, chimeric, or foreign in nature.Typically a genetic construct will comprise a “coding sequence”. A“coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence.

“Promoter” or “Initiation control regions” refers to a DNA sequencecapable of controlling the expression of a coding sequence or functionalRNA. In general, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a gene to be expressed in most celltypes at most times are commonly referred to as “constitutivepromoters”.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from agene. Expression may also refer to translation of mRNA into apolypeptide. “Antisense inhibition” refers to the production ofantisense RNA transcripts capable of suppressing the expression of thetarget protein. “Overexpression” refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. “Co-suppression” refers to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020).

The term “Messenger RNA (mRNA)” as used herein, refers to the RNA thatis without introns and that can be translated into protein by the cell.

The term “non-functional gene” as used herein refers to a gene that doesnot express the encoded protein normally as in the wild type strainwhere the gene is endogenous. Expression of a non-functional gene may bedisrupted at any level, such as transcription, RNA processing, ortranslation. A non-functional gene typically has little or no expressionof the encoded protein. However it may also code for a modified proteinthat has lower enzyme activity than the wild type protein.

The term “transformation” as used herein, refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. The transferred nucleic acid may be in the form of aplasmid maintained in the host cell, or some transferred nucleic acidmay be integrated into the genome of the host cell. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,wherein the effect is used to track the inheritance of a nucleic acid ofinterest and/or to identify a cell or organism that has inherited thenucleic acid of interest.

The terms “substantially eliminated” xylitol production and“substantially no” by-product xylitol refer to the case where the amountof xylitol detected using typical laboratory analysis is close to zero.

The term “high concentration of mixed sugars” refers to a total sugarconcentration in the medium that results in inhibition of growth of GFORmutant xylose-utilizing Z. mobilis. This is typically greater than about100 g/L, although the exact concentration may vary depending on othercomponents in the medium.

The term “fermentable sugar” refers to oligosaccharides andmonosaccharides that can be used as a carbon source by a microorganismin a fermentation process.

The term “lignocellulosic” refers to a composition comprising bothlignin and cellulose. Lignocellulosic material may also comprisehemicellulose.

The term “cellulosic” refers to a composition comprising cellulose andadditional components, including hemicellulose.

The term “saccharification” refers to the production of fermentablesugars from polysaccharides.

The term “pretreated biomass” means biomass that has been subjected topretreatment prior to saccharification.

“Biomass” refers to any cellulosic or lignocellulosic material andincludes materials comprising cellulose, and optionally furthercomprising hemicellulose, lignin, starch, oligosaccharides and/ormonosaccharides. Biomass may also comprise additional components, suchas protein and/or lipid. Biomass may be derived from a single source, orbiomass can comprise a mixture derived from more than one source; forexample, biomass could comprise a mixture of corn cobs and corn stover,or a mixture of grass and leaves. Biomass includes, but is not limitedto, bioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, sludge from paper manufacture, yard waste, woodand forestry waste. Examples of biomass include, but are not limited to,corn grain, corn cobs, crop residues such as corn husks, corn stover,grasses, wheat, wheat straw, barley, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers andanimal manure.

“Biomass hydrolysate” refers to the product resulting fromsaccharification of biomass. The biomass may also be pretreated prior tosaccharification.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989(hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. andEnquist, L. W. Experiments with Gene Fusions; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. etal., In Current Protocols in Molecular Biology, published by GreenePublishing and Wiley-Interscience, 1987.

The present invention relates to a process for producing ethanol fromxylose-containing medium using GFOR mutants of xylose-utilizingZymomonas. The engineered strains of xylose-utilizing Zymomonas withreduced GFOR enzyme activity have enhanced ethanol production. Achallenge for improving ethanol production by xylose-utilizing Z mobilisis reducing or eliminating the synthesis of xylitol, which (a)represents a non-value adding carbon sink; (b) inhibits the first stepof xylose utilization; and (c) is phosphorylated to a toxic deadendintermediate that inhibits bacterial growth. Applicants have discoveredthat the endogenous enzyme GFOR is predominantly responsible for xylitolsynthesis in vivo and that by reducing or eliminating GFOR enzymeactivity, ethanol production (rate, yield and titer) from xylose isimproved.

Xylose-Utilizing Zymomonas Host Strain

Any strain of Zymomonas that is able to utilize xylose as a carbonsource may be used as a host for preparing the strains used in thepresent process. Strains of Z. mobilis that have been engineered forxylose fermentation to ethanol are particularly useful. Endogenous genesmay provide part of the metabolic pathway, or may be altered by anyknown genetic manipulation technique to provide a protein with enzymeactivity useful for xylose metabolism. For example, the endogenoustransketolase may complement other introduced enzyme activities increating a xylose utilization pathway. Typically four genes have beenintroduced into Z mobilis for expression of four enzymes involved inxylose metabolism (FIG. 1) as described in U.S. Pat. No. 5,514,583,which is herein incorporated by reference. These include genes encodingxylose isomerase, which catalyzes the conversion of xylose to xyluloseand xylulokinase, which phosphorylates xylulose to form xylulose5-phosphate. In addition, transketolase and transaldolase, two enzymesof the pentose phosphate pathway, convert xylulose 5-phosphate tointermediates that couple pentose metabolism to the glycolyticEntner-Douderoff pathway permitting the metabolism of xylose to ethanol.DNA sequences encoding these enzymes may be obtained from any ofnumerous microorganisms that are able to metabolize xylose, such asenteric bacteria, and some yeasts and fungi. Sources for the codingregions include Xanthomonas, Klebsiella, Escherichia, Rhodobacter,Flavobacterium, Acetobacter, Gluconobacter, Rhizobium, Agrobacterium,Salmonella, Pseudomonads, and Zymomonas. Particularly useful are thecoding regions of E. coli.

The encoding DNA sequences are operably linked to promoters that areexpressed in Z. mobilis cells such as the promoters of Z. mobilisglyceraldehyde-3-phosphate dehydrogenase (GAP promoter), and Z. mobilisenolase (ENO promoter). The coding regions may individually be expressedfrom promoters, or two or more coding regions may be joined in an operonwith expression from the same promoter. The resulting chimeric genes maybe introduced into Zymomonas and maintained on a plasmid, or integratedinto the genome using, for example, homologous recombination,site-directed integration, or random integration. Xylose-utilizingstrains that are of particular use include CP4(pZB5) (U.S. Pat. No.5,514,583), ATCC31821/pZB5 (U.S. Pat. No. 6,566,107), 8b (US20030162271; Mohagheghi et al., (2004) Biotechnol. Lett. 25; 321-325),and ZW658 (described herein; deposited, ATTCC # PTA-7858).

Zymomonas strains that are additionally engineered to utilize sugarsother than xylose, which it does not naturally use, may be used in thepresent process. An example is a strain of Z. mobilis engineered forarabinose utilization as described in U.S. Pat. No. 5,843,760, which isherein incorporated by reference.

Discovery of Xylitol Synthesis by GFOR

Synthesis of the unwanted by-product xylitol by xylose-utilizing strainsof Z. mobilis reduces the yield of ethanol and results in the formationof xylitol 5-phosphate which is a toxic compound that inhibits bacterialgrowth (see FIG. 2). In addition, xylitol is a potent inhibitor ofxylose isomerase, the first enzyme in the engineered pathway for xyloseutilization, and its synthesis reduces the ability of the cells tometabolize xylose. Although in vitro experiments have established thatthere are at least two pathways for xylitol formation in Z. mobilis(Feldmann et al. supra, Danielson et al. supra), applicants havediscovered that the majority of xylitol that is produced physiologicallyis the result of GFOR enzyme activity. Applicants have found that theamount of xylitol that is synthesized by Z. mobilis strains that canutilize xylose (or xylulose synthesizing derivatives of wild type Z.mobilis) that are grown on xylose-containing media is greatly reduced inthe absence of GFOR enzyme activity. Applicants also found thatconversion of xylose to xylulose is a prerequisite for GFOR-mediatedxylitol production in vivo, and that this reaction can only occur in Z.mobilis strains that express xylose isomerase. Thus it is proposed thatthe major physiological source of xylitol in Z. mobilis strains that areengineered to grow on xylose is synthesized by GFOR via one or both ofthe reactions that are depicted in Diagrams II and III.

Note that in both schemes xylulose serves as the obligatory electronacceptor for GFOR and that this compound is reduced to xylitol incontrast to the known reaction with fructose that results in sorbitolproduction (Diagram I). Although GFOR is quite specific for glucose andfructose, it has been shown that it can use other sugars as electrondonors and electron acceptors, albeit rather poorly (Zachariou andScopes (1986) Journal of Bacteriology 167:863-869). Thus, when xyloseand fructose were incubated with the purified protein, sorbitolproduction was observed but there was about a 12-fold reduction in GFORenzyme activity compared to the control reaction with glucose. In thesame paper it was shown that xylulose can substitute for fructose as anelectron acceptor, and that this reaction gives rise to xylitol asdepicted in Diagram III. However, with this combination of substratesthere was about a 14-fold decrease in GFOR enzyme activity. In additionto these observations, it has also been shown that cell-free extractsprepared from wildtype Z. mobilis are able to generate xylitol fromxylose when purified xylose isomerase is added to the reaction mixtureto provide a source of xylulose (Danielson supra), thus demonstratingthat GFOR can also catalyze the reaction that is depicted in Diagram II.However, whether or not these GFOR-mediated reactions occur in livingcells and, if they do, to what extent they contribute to xylitolformation in vivo remained to be determined prior to applicants'discovery. The same uncertainties pertained to the NADPH-dependentaldose reductase activity that is also present in wild type Z. mobiliscell-free extracts, that is able to directly convert xylose to xylitol(Feldmann et al. supra). Indeed, none of the experiments with cell-freeextracts noted above provided any insight on the relative contributionsof GFOR and NADPH-dependent aldose reducatase to xylitol formation invitro, let alone in vivo under process relevant conditions. Thusapplicants' finding that GFOR is principally responsible for xylitolproduction in Z. mobilis strains that are engineered to grow on xyloseunder physiological conditions in xylose containing media was surprisingand could not be anticipated from prior art.

Altering GFOR Gene Expression

A xylose-utilizing Zymomonas strain used in the present process isengineered such that there is reduced or no expression of the GFORencoding gene, so that xylitol synthesis is reduced. Any geneticmodification method known by one skilled in the art for reducing thepresence of a functional enzyme may be used to alter GFOR expression.Methods include, but are not limited to, deletion of the entire gene ora portion of the gene encoding GFOR, inserting a DNA fragment into theGFOR gene (in either the promoter or coding region) so that the proteinis not expressed or is expressed at lower levels, introducing a mutationinto the GFOR coding region which adds a stop codon or frame shift suchthat a functional protein is not expressed, and introducing one or moremutations into the GFOR coding region to alter amino acids so that anon-functional or a less enzymatically active protein is expressed. Inaddition, GFOR expression may be blocked by expression of an antisenseRNA or an interfering RNA, and constructs may be introduced that resultin cosuppression. All of these methods may be readily practiced by oneskilled in the art making use of the known sequence encoding GFOR (SEQID NO:38). DNA sequences surrounding the GFOR coding sequence are alsouseful in some modification procedures and are available for Z. mobilisin the complete genome sequence (GenBank Accession #AE008692).

A particularly suitable method for creating a genetically modified GFORstrain, as exemplified herein in Examples 3 and 5, is using homologousrecombination mediated by GFOR flanking DNA sequences bounding aspectinomycin resistance gene or other selectable marker, leading toinsertion of the selectable marker in the GFOR coding region such that afunctional GFOR enzyme is not expressed. In addition, the selectablemarker may be bounded by site-specific recombination sites, so thatfollowing expression of the corresponding site-specific recombinase, theresistance gene is excised from the GFOR gene without reactivating thelatter. The site-specific recombination leaves behind a recombinationsite which disrupts expression of the GFOR enzyme. The homologousrecombination vector may be constructed to also leave a deletion in theGFOR gene following excision of the selectable marker, as is well knownto one skilled in the art.

It is preferred to completely eliminate the expression of GFOR, howeveruse of recombinant Zymomonas strains with greatly reduced expression ofGFOR is also an embodiment of the present process. In this case, anon-functional GFOR gene refers to not functioning in the normal mannersuch that lower than normal levels of GFOR enzyme are present. Somemethods of gene inactivation may result in some remaining low-levelexpression, such as co-suppression. Herein, a modified GFOR strainrefers to a genetically modified strain with reduced or no GFOR enzymeactivity.

Growth and Ethanol Production by GFOR Modified Strain

A GFOR modified xylose-utilizing Zymomonas strain used in the presentprocess is grown in a medium containing xylose in the absence orpresence of other sugars (“mixed sugars”). The mixed sugars include atleast one additional sugar to xylose. Any sugar that may provide anenergy source for metabolism of the Zymomonas cells, or any sugar thatis present in a mixture containing xylose may be included. It isdesirable to grow GFOR modified xylose-utilizing Z. mobilis cells onsugars that are produced from biomass saccharification. Typicallybiomass is pretreated, for example as described in Patent ApplicationWO2004/081185 and in co-owned and co-pending U.S. application60/670,437, and then treated with saccharification enzymes as reviewedin Lynd, L. R., et al. (Microbiol. Mol. Biol. Rev. (2002) 66:506-577).Biomass saccharification produces sugars that may typically include amixture of xylose with glucose, fructose, sucrose, galactose, mannose,and/or arabinose. Preferred is a mixed sugars composition that includesxylose and glucose, where additional sugars may be present.

The ratio of different sugars may vary in the mixture, with xylosetypically at least about 10% of the total amount of sugars. Preferablyxylose is between about 40% and about 60%. Fructose is present in sugarsproduced by saccharification of some biomass such as sugar cane bagasse,and may replace a portion of xylose or glucose, such that xylose remainsat least about 10% of the sugar mixture. In addition, arabinose isderived from hemicellulose and thus is a typical component of mixedsugars derived from saccharified biomass containing hemicellulose.

Under fermentation conditions where xylitol would not be produced by axylose-utilizing Z. mobilis strain that is not a GFOR modified strain,GFOR modified xylose-utilizing Z. mobilis strains grow and produceethanol comparably to non-GFOR modified strains. For example, in lowsugar medium, such as at about 100 g/L mixed sugars with a 5:4 ratio ofglucose to xylose, the GFOR modified xylose-utilizing Z. mobilis cellsperform similarly to non-GFOR modified strains.

For maximal ethanol production and efficiency of fermentation it isdesirable to grow a xylose-utilizing ethanologen in medium containinghigh levels of sugars, including xylose. The mixed sugars may be used ina high concentration in medium for growth of GFOR modifiedxylose-utilizing Z. mobilis strains in the present process. This allowsthe direct use of biomass saccharification sugars, or use with littledilution, thereby reducing fermentation volumes, which is desirable forcommercial scale ethanol production. High sugars concentrations are usedso that greater concentrations of ethanol may be produced. The mixedsugars concentration in the fermentation medium is typically at leastabout 120 g/L and up to about 300 g/L. Particularly useful is a highconcentration of mixed sugars that is between about 150 g/L and about235 g/L.

In the high concentration mixed sugars conditions desired for productionof ethanol, sorbitol is included in the fermentation medium for the GFORmodified xylose-utilizing Z. mobilis. Applicants surprisingly found thataddition of sorbitol to the high mixed sugars medium allowed good growthof GFOR modified xylose-utilizing Z. mobilis, whereas without inclusionof sorbitol the GFOR modified xylose-utilizing Z. mobilis showed littleor no growth. This is in marked contrast to GFOR producing strains thatare able to adapt to the concentrated sugar mixture without sorbitoladdition after a 12-36 hour lag period. In medium lacking fructose orsucrose (as a source of fructose), it was not expected that GFOR wouldsynthesize sorbitol or play a role in osmotic adaptation. With nofructose present in the growth medium, the known reaction of GFOR forsorbitol synthesis, shown in Diagram I, could not proceed. The abilityof xylose-utilizing Z. mobilis strains with normal GFOR enzyme activityto grow in a concentrated mixture of glucose and xylose, albeit with along lag period, suggested that sorbitol synthesis by GFOR was notneeded for osmotic adaptation, since without fructose GFOR would not beexpected to synthesize sorbitol. Thus eliminating GFOR enzyme activitywas not expected to have an effect on the level of sorbitol productionin growth medium that lacks fructose, and a sorbitol requirement forgrowth of the GFOR modified xylose-utilizing Z. mobilis strain in aconcentrated mixture of glucose and xylose was completely unexpected.

Sorbitol (D-sorbitol and/or L-sorbitol) may be present in the medium atconcentrations that are between about 0.5 mM and 200 mM. More suitablefinal concentrations in the medium are concentrations between about 2 mMand 100 mM, with concentrations between 5 mM and 20 mM preferred.Mannitol may be used in the medium instead of sorbitol, or incombination with sorbitol. Mannitol was found to have similar effects tothose of sorbitol in co-owned and co-pending U.S. application No.60/847,997, which is herein incorporated by reference. In addition, itwas found that galactitol and/or ribitol may be used in place of or incombination with sorbitol and/or mannitol. Sorbitol, mannitol,galactitol, ribitol or combinations thereof are all used in the sameconcentrations as described for sorbitol. Under fermentation conditionswhere xylitol would be produced by a xylose-utilizing Z. mobilis strainthat is not a GFOR modified strain, such as in high sugar medium in thepresence or absence of inhibitors such as acetate, GFOR modifiedxylose-utilizing Z. mobilis strains used in the process outperformnon-GFOR modified strains. Applicants found that both the total amountof xylose that is consumed and the final ethanol titer are greater for aGFOR modified strain than a non-modified strain. Furthermore, no xylitolwas produced in fermentations by GFOR modified xylose-utilizing Z.mobilis under process-relevant conditions, although small amounts couldbe synthesized by a non-GFOR mechanism under certain circumstances asshown in Example 6 herein.

The improvement in xylose utilization and ethanol production variesunder different fermentation conditions. Under conditions where a higherlevel of xylitol is produced by a GFOR non-modified xylose-utilizing Z.mobilis strain, the lack of xylitol synthesis leads to a greater effectof the GFOR mutation. For example, when an inhibitor such as acetate ispresent in the medium, larger amounts of xylitol are produced by GFORnon-modified strains. This xylitol production is completely eliminatedby the GFOR mutation allowing a greater increase in xylose utilizationand ethanol production than in conditions where low amounts of xylitolwould have been produced without the GFOR mutation. Since acetate istypically present in treated cellulosic biomass, reduced sensitivity toacetate is desired in an ethanologen to be grown on carbon sourcesderived from treated cellulosic biomass. Thus fermentation using a GFORmodified xylose-utilizing Z. mobilis strain is particularly beneficialwhen biomass hydrolysate is used in fermentation.

Fermentation for Ethanol Production

For production of ethanol, recombinant GFOR modified xylose-utilizing Z.mobilis is brought in contact with medium that contains mixed sugarsincluding xylose. When the mixed sugars concentration is high such thatgrowth is inhibited, the medium includes sorbitol, mannitol, or amixture thereof. Galactitol or ribitol may replace or be combined withsorbitol or mannitol. The Z. mobilis grows in the medium wherefermentation occurs and ethanol is produced. The fermentation is runwithout supplemented air, oxygen, or other gases (which may includeconditions such as anaerobic, microaerobic, or microaerophilicfermentation), for at least about 24 hours, and may be run for 30 ormore hours. The timing to reach maximal ethanol production is variable,depending on the fermentation conditions. Typically, if inhibitors arepresent in the medium, a longer fermentation period is required. Thefermentations may be run at temperatures that are between about 30° C.and about 37° C., at a pH of about 4.5 to about 7.5.

In the present process GFOR modified xylose-utilizing Z. mobilis may begrown in medium containing mixed sugars including xylose in laboratoryscale fermenters, and in scaled up fermentation where commercialquantities of ethanol are produced. Where commercial production ofethanol is desired, a variety of culture methodologies may be applied.For example, large-scale production from GFOR modified xylose-utilizingZ. mobilis may be produced by both batch and continuous culturemethodologies. A classical batch culturing method is a closed systemwhere the composition of the medium is set at the beginning of theculture and not subjected to artificial alterations during the culturingprocess. Thus, at the beginning of the culturing process the medium isinoculated with the desired organism and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present process andcomprise a typical batch system with the exception that the substrate isadded in increments as the culture progresses. Fed-Batch systems areuseful when catabolite repression is apt to inhibit the metabolism ofthe cells and where it is desirable to have limited amounts of substratein the medium. Measurement of the actual substrate concentration inFed-Batch systems is difficult and is therefore estimated on the basisof the changes of measurable factors such as pH and the partial pressureof waste gases such as CO₂. Batch and Fed-Batch culturing methods arecommon and well known in the art and examples may be found inBiotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger,and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland,Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227,(1992), herein incorporated by reference.

Commercial production of ethanol may also be accomplished with acontinuous culture. Continuous cultures are open systems where a definedculture medium is added continuously to a bioreactor and an equal amountof conditioned medium is removed simultaneously for processing.Continuous cultures generally maintain the cells at a constant highliquid phase density where cells are primarily in log phase growth.Alternatively, continuous culture may be practiced with immobilizedcells where carbon and nutrients are continuously added, and valuableproducts, by-products or waste products are continuously removed fromthe cell mass. Cell immobilization may be performed using a wide rangeof solid supports composed of natural and/or synthetic materials as isknown to one skilled in the art.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by medium turbidity, is kept constant.Continuous systems strive to maintain steady state growth conditions andthus the cell loss due to medium being drawn off must be balancedagainst the cell growth rate in the culture. Methods of modulatingnutrients and growth factors for continuous culture processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, supra.

Particularly suitable for ethanol production is a fermentation regime asfollows. The desired GFOR modified xylose-utilizing Z. mobilis strain isgrown in shake flasks in semi-complex medium at about 30° C. to about37° C. with shaking at about 150 rpm in orbital shakers and thentransferred to a 10 L seed fermentor containing similar medium. The seedculture is grown in the seed fermentor anaerobically until OD₆₀₀ isbetween 3 and 6, when it is transferred to the production fermentorwhere the fermentation parameters are optimized for ethanol production.Typical inoculum volumes transferred from the seed tank to theproduction tank range from about 2% to about 20% v/v. Typicalfermentation medium contains minimal medium components such as potassiumphosphate (1.0-10.0 g/l), ammonium sulfate (0-2.0 g/l), magnesiumsulfate (0-5.0 g/l), a complex nitrogen source such as yeast extract orsoy based products (0-10 g/l). A final concentration of about 5 mMsorbitol or mannitol is present in the medium. Mixed sugars includingxylose and at least one additional sugar such as glucose (or sucrose),providing a carbon source, are continually added to the fermentationvessel on depletion of the initial batched carbon source (50-200 g/l) tomaximize ethanol rate and titer. Carbon source feed rates are adjusteddynamically to ensure that the culture is not accumulating glucose inexcess, which could lead to build up of toxic byproducts such as aceticacid. In order to maximize yield of ethanol produced from substrateutilized, biomass growth is restricted by the amount of phosphate thatis either batched initially or that is fed during the course of thefermentation. The fermentation is controlled at pH 5.0-6.0 using causticsolution (such as ammonium hydroxide, potassium hydroxide, or sodiumhydroxide) and either sulfuric or phosphoric acid. The temperature ofthe fermentor is controlled at 30° C.-35° C. In order to minimizefoaming, antifoam agents (any class-silicone based, organic based etc)are added to the vessel as needed. An antibiotic, for which there is anantibiotic resistant marker in the strain, such as kanamycin, may beused optionally to minimize contamination.

Any set of conditions described above, and additionally variations inthese conditions that are well known to one skilled in the art, aresuitable conditions for production of ethanol by a xylose-utilizingrecombinant Zymomonas strain.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. andEnquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. etal., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).

The meaning of abbreviations is as follows: “kb” means kilobase(s), “bp”means base pairs, “nt” means nucleotide(s), “hr” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “L” meansliter(s), “ml” means milliliter(s), “μL” means microliter(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “mM” means millimolar, “μM” meansmicromolar, “nm” means nanometer(s), “μmol” means micromole(s), “pmol”means picomole(s), “Cm” means chloramphenicol, “Cm^(r)” meanschloramphenicol resistant, “Cm^(s)” means chloramphenicol sensitive,“Sp^(r)” means spectinomycin resistance, “Sp^(s)” means spectinomycinsensitive, “XI” is xylose isomerase, “XK” is xylulokinase, “TAL” istransaldolase, “TKT” is transketolase, “EFT” means elapsed fermentationtime, “RM” means rich medium containing 10 g/L yeast extract plus 2 g/LKH₂PO₄, “MM” means mating medium containing 10 g/L yeast extract, 5 g/Ltryptone, 2.5 g/L (NH₄)₂SO₄ and 0.2 g/L KH₂PO₄.

Preparation of Cell-Free Extracts of Zymomonas for Enzymatic Assays

Cells were grown in 50 ml of RM+2% glucose at 30° C. overnight to anOD₆₀₀ of 1.0-1.2. Cells were harvested by centrifugation at 4500 rpm for10 min at 4° C. The supernatant was discarded and the cell pellet washedwith 25 ml ice-cold sonication buffer. (10 mM Tris, pH 7.6, 10 mMMgCl₂), followed by centrifugation at 4500 rpm for 10 min. The pelletwas resuspended in 2.0-2.5 ml sonication buffer plus 1 mMdithiothreitol. A 500 μl aliquot was centrifuged for 1 min in aneppendorf centrifuge at 4° C. Most of supernatant was discarded, leaving˜10-20 μl behind to keep the pellet from drying out. The cells werefrozen and stored at −80° C. until assayed. Prior to assay, the cellswere thawed and resuspended with 500 μl of sonication buffer plus 1 mMdithiothreitol. The mix was sonicated 2× for 45 seconds at 62% dutycycle and an output control of 2 using a Branson sonifier 450, lettingsamples cool ˜3-5 min between sonications. Samples were centrifuged at14,000 rpm for 60 min in a Beckman microfuge at 4° C. The supernatantwas transferred to a new tube and kept at 4° C. The Pierce BCA assay wasused for determining protein concentrations.

The transketolase (TKT) assay was usually performed first since thisenzyme is more labile than the others. A diagram of the TKT assay isshown in FIG. 3A.

In a microplate assay, 20 μl of cell free extract was added to each wellin a reaction mix, at 30° C., that included the following finalconcentrations of components: 0.37 mM NADP, 50 mM Tris HCl pH 7.5, 8.4mM Mg Cl₂, 0.1 mM TPP ((thiamine pyrophosphate chloride), 0.6 mM E4P(erythrose-4-phosphate), 4 mM BHP (betahydroxypyruvate), 4 U/ml PGI(phosphoglucose isomerase), and 4 U/ml G6PD (glucose-6-phosphatedehydrogenase). The A₃₄₀ was read on a plate reader for 3-5 min. TKTactivity was calculated as follows:

1 unit corresponds to the formation of 1 μmol of D-fructose6-phosphate/min at 30° C. U (μmole/min) = slope (dA₃₄₀/min) * volume ofreaction (μL)/6220/0.55 cm (moles of NADP→NADPH is 6220 A₃₄₀ per moleper L in a 1 cm cuvette) (pathlength of 200 μl per well in microplate =0.55 cm) Specific Activity (μmole/min-mg) = μmole/min/proteinconcentration (mg)

The basis of the transaldolase (TAL) assay is shown in FIG. 3B. In amicroplate assay, 20 μl of cell free extract was added to each well in areaction mix, at 30° C., that included the following finalconcentrations of components: 0.38 mM NADH, 87 mM thiethanolamine, 17 mMEDTA, 33 mM F6P (fructose-6-phosphate), 1.2 mM E4P(erythrose-4-phosphate), 2.0 U/ml GDH (Glycerol-3-phosphatedehydrogenase), and 20 U/ml TPI (Triose phosphate isomerase). The platewas incubated for 5 min., then the A₃₄₀ was read for 3-5 min. TALactivity was calculated as follows: 1 unit corresponds to the formationof 1 μmol of D-glyceraldehyde per minute at 30° C.

U (μmole/min) = slope (dA₃₄₀/min) * volume of reaction (μL)/6220/0.55 cm(moles of NADH→NAD is 6220 A₃₄₀ per mole per L in a 1 cm cuvette)(pathlength of 200 ul per well in microplate = 0.55 cm) SpecificActivity (μmole/min-mg) = μmole/min/protein The basis of the xyloseisomerase (XI) assay is shown in FIG. 3C.In a microplate assay, 20 μl of cell free extract was added to each wellin a reaction mix, at 30° C., that included the following finalconcentrations of components: 0.256 mM NADH, 50 mM xylose, 10 mM MgSO₄,10 mM thiethanolamine, and 1 U/ml SDH (sorbitol dehydrogenase). The A₃₄₀was read on a plate reader for 3-5 min. XI activity was calculated asfollows: 1 unit of XI corresponds to the formation of 1 μmole ofD-xylulose per minute at 30° C.

U (μmole/min) = slope (dA₃₄₀/min) * volume of reaction (μL)/6220/0.55 cm(moles of NADHP→NAD is 6220 A₃₄₀ per mole per L in a 1 cm cuvette)(pathlength of 200 μl per well in microplate = 0.55 cm) SpecificActivity (μmole/min − mg) = μmole/min/protein concentration (mg)

The basis of the xylulokinase (XK) assay is shown in FIG. 3D. In amicroplate assay, 20 μl of cell free extract was added to each well in areaction mix, at 30° C., that included the following finalconcentrations of components: 0.2 mM NADH, 50 mM Tris HCl pH 7.5, 2.0 mmMgCl₂-6H₂O, 2.0 M ATP 0.2 M PEP (phosphoenolpyruvate), 8.5 mMD-xylulose, 5 U/ml PK (pyruvate kinase), and 5 U/ml LDH (lactatedehydrognase). The A₃₄₀ was read on a plate reader for 3-5 min. XIactivity was calculated as follows:

1 unit corresponds to the formation of 1 μmole of D-xylulose toD-xylulose-5-phosphate per minute at 30° C. U (μmole/min) = slope(dA₃₄₀/min) * volume of reaction (μL)/6220/0.55 cm (moles of NADH→NAD is6220 A₃₄₀ per mole per L in a 1 cm cuvette) (pathlength of 200 μl perwell in microplate = 0.55 cm) Specific Activity (μmole/min-mg) =μmole/min/protein concentration (mg)

HPLC Method

The analysis was done with an Agilent 1100 series HPLC and AgilentChemStation software for LC 3D. The column was BioRad Aminex HPX-87H(HPLC Organic Analysis Column 125-0140) with BioRad Micro-GuardCartridge Cation-H (125-0129). The operating conditions were:

Flow 0.6 mL/min Solvent 0.01N H₂SO₄ Stop Time 25 min Injection Volume 5μl Auto Sampler Temp Control @ 10° C. or 4° C. Column Temp 55° C.Detector Refractive Index (40° C.) with External Standard CalibrationCurves

Example 1 Construction of ZW658, a Xylose-Fermenting Zymomonas mobilisStrain

ZW658 was constructed by integrating two operons, P_(gap)xylAB andP_(gap)taltkt, containing four xylose-utilizing genes encoding xyloseisomerase, xylulokinase, transaldolase and transketolase, into thegenome of ZW1 (ATCC #31821) via sequential transposition events, andfollowed by adaptation on selective media containing xylose. Previously,a xylose-fermenting Zymomonas mobilis strain called 8b was constructed,as described in United States Patent Application 20030162271, byintegrating the two operons P_(gap)xylAxylB and P_(eno)taltkt, alongwith selectable antibiotic markers, into the genome of Zymomonas mobilis5C via a combination of homologous recombination and transposonapproaches followed by adaptation and NTG mutagenesis. In thepreparation of ZW658, transposition (Epicentre's EZ::Tn in vitrotransposition system) was used, as opposed to site specific homologousrecombination, because this approach offers the advantages of multiplechoices of integration sites and relatively high insertion frequency.The four genes encoding the xylose utilization enzymes were arranged andcloned as two separate operons: P_(gap)xylAB and P_(gap)taltkt for theintegration. An antibiotic resistance marker, a chloramphenicolresistance (Cm^(r)) gene flanked by two P1 phage Cre-recombinaserecognition sequences (loxP), was attached to each operon for theselection of integrants. The integration of the two operons wasaccomplished in a two-step, sequential manner: P_(gap)taltkt followed byP_(gap)xylAB. Cm resistance selection was used in both integrationevents, since it was removed by expressing a Cre recombinase on aplasmid followed by curing of the plasmid after each integration. Thisprocess allowed the use of the same antibiotic marker for selectionmultiple times. More importantly, it allowed the removal of theantibiotic marker introduced for selection of the integration of theoperons. This process eliminated the negative impact of antibioticresistance gene(s) on the fermentation strain for commercial use.

Construction of pMODP_(gap)taltktCm for Transposition

As described in the US Patent Application 20030162271 (Example 9therein), a 2.2 kb DNA fragment containing the transketolase (tkt)coding region from E. coli was isolated from pUCtaltkt (US PatentApplication 20030162271) by BgIII/XbaI digestion and cloned in a PMOD(Epicentre Biotechnologies, Madison, Wis.) vector digested withBamHI/XbaI, resulting in pMODtkt. A PCR fragment named P_(gap)tal wasgenerated by fusing the promoter region of the Zymomonas mobilis gap(P_(gap); glyceraldehyde-3-phosphate dehydrogenase) gene to the codingregion of E. coli transaldolase (tal) as follows. A P_(gap) fragment wasamplified from pZB4, the construction of which is described in U.S. Pat.No. 5,514,583 (Example 3), using primers with SEQ ID NOs:1 and 2. pZB4contains a P_(gap)-xylA/xylB operon and a P_(ENO)-tal/tkt operon. A talcoding region fragment was amplified from pZB4 using primers with SEQ IDNOs: 3 and 4. A P_(gap)tal fragment was amplified using the P_(gap) andtal fragments as template using primers with SEQ ID NOs:5 and 6. Thisfragment was digested with XbaI and cloned into the plasmid pMODtkt,upstream of the tkt coding region. A loxP::Cm fragment was generated byPCR using Cmlox(F,sfi) and Cmlox(R,sfi) primers (SEQ ID NOs:7 and 8) andpZB186 as the template. pZB186 is a combination of a native Z. mobilisplasmid and pACYC184, described in U.S. Pat. No. 514,583 (Example 3) andZhang et al. ((1995) Science 267:240-243). Finally, the loxP::Cm PCRfragment was inserted in the SfiI site of the plasmid containingP_(gap)taltkt to form the integrative plasmid pMODP_(gap)taltktCm (FIG.4). In this plasmid, the P_(gap)taltkt loxP::Cm fragment was insertedbetween two mosaic ends (transposase binding sites) in the PMOD vector.The complete nucleotide sequence for the pMODP_(gap)taltktCm plasmid isgiven as SEQ ID NO:9.

Transposition and Transformation of pMODP_(gap)taltktCm in ZW1

Plasmid PMOD is a pUC-based vector, and therefore is a non-replicativevector in Zymomonas. Plasmid pMODP_(gap)taltktCm was treated withtransposase in the presence of Mg²⁺ at room temperature for one hour andused to transform ZW1 cells by electroporation (using a BioRad GenePulser set at 200 ohms, 25 μF and 16 kV/cm). Electroporated cells wereincubated in a mating medium (MM), which consists of 10 g/L yeastextract, 5 g/L tryptone, 2.5 g/L (NH₄)₂SO₄, 0.2 g/L K₂HPO₄) supplementedwith 50 g/L glucose and 1 mM MgSO₄ for 6 hours at 30° C. Thetransformation mixture was plated on agar plates containing 15 g/L Bactoagar in MM supplemented with 50 g/L glucose and 120 μg/mLchloramphenicol and incubated anerobically at 30° C. The transformantswere visible after about 2 days. The transformation/transpositionfrequency was approx. 3×10¹/μg DNA.

A total of 39 Cm^(r) transformant colonies was obtained. Twenty-onecolonies were picked and further analyzed by PCR and enzymatic activityassays. PCR using primers SEQ ID NOs:10 and 11 confirmed the presence ofa 3 kb DNA fragment containing tal and tkt coding regions in thetransformants. Back transformation with plasmid DNA from the 21integrant colonies generated no back transformants in E. coli suggestingthe tal and tkt were integrated in the genome of ZW1. These integrantswere tested for transaldolase and transketolase activities usingprotocols modified for microplates (General Methods). The Pierce BCAprotein assay was used for the determination of protein concentrations.The transformants were grown up in RM medium containing 2% (w/v) glucosesupplemented with 120 μg/ml chloramphenicol) in 50 ml conical centrifugetubes at 30° C. The control strains 8b and ZW1 were grown up as well (RMplus 2% glucose was used for ZW1) for enzymatic assays. Cells wereharvested when the OD₆₀₀ reached 1.0. Cells were washed once andresuspended in sonication buffer (10 mM Tris-HCl, pH 7.6 and 10 mMMgCl₂). Enzymatic assays were conducted as described in US PatentApplication, 20030162271. Units are given as μmole/min-mg. All sampleshad transaldolase and transketolase activities except for one.

Southern hybridization was performed on genomic and plasmid DNA ofselected integrants digested with PstI using a tkt probe. ZW1 DNA didnot hybridize with the tkt probe. A common 1.5 kb band was visible inall integrant genomic DNA samples, which is the expected DNA fragmentbetween a PstI site in tkt and a PstI site in tal. A second visible highmolecular weight (6 kb or greater) band was unique between independentlines T2, T3, T4 and T5 indicating a separate genomic integration sitein each line. Interestingly, both plasmid and genomic DNA of T5hybridized with the tkt probe indicating it was likely thatP_(gap)taltkt was also integrated in T5 on the native plasmid. Thesefour strains (T2, T3, T4 and T5) were selected for further Cre treatmentto remove the Cm^(r) marker.

Cre Treatment to Remove Cm^(r) Marker from Taltkt Integrants

To remove the Cm^(r) marker from the chromosome, T2, T3, T4 and T5 weretransformed with pZB188/Spec-Cre. This plasmid is a derivative of theZymomonas-E. coli shuttle vector pZB188 [Zhang et al. (1995) Science267:240-243; U.S. Pat. No. 5,514,583] that contains an expressioncassette for Cre Recombinase. pZB188/Spec-Cre is identical to the CreExpression vector that is described In Example 10 (pZB188/Kan-Cre),except that it has a spectinomycin-resistance gene instead of akanamycin-resistance gene. The transformants were selected on MM agarplates supplemented with 2% glucose and 200 μg/ml spectinomycin). Sp^(r)resistant colonies were picked onto RM agar plates supplemented with 2%glucose and 200 μg/ml spectinomycin and RM agar plates supplemented with2% glucose and 120 μg/mL Cm. One hundred percent of the colonies pickedwere Cm^(s) indicating the high efficiency excision of Cm^(r) by Cre.Sp^(r)Cm^(s) transformants were cultured in RM plus 2% glucose at 37° C.for 2 to 5 daily transfers to cure pZB188aadACreF. At each transfer,cells were diluted and plated on RM plus 2% glucose agar plates forpicking onto additional plates of the same medium with or without 200μg/mL Sp. Sp^(s) colonies were analyzed by PCR to confirm the loss ofpZB188aadACreF. The plasmid-cured descendents of the integrants werenamed T2C, T3C, T4C and T5C. To examine whether these transpositionintegrants were stable, these 4 strains were grown in RM plus 2% glucoseand then transferred to 10 ml of the same medium and grown at 37° C. induplicate test tubes. Cells were transferred daily for ten days, orapproximately 100 generations. Colonies were diluted and plated onto RMGplates for colony isolation after the 1st and 10th transfers. Twelvecolonies from each transfer of each strain tested positive for thepresence of P_(gap)taltkt by colony PCR using 5′ P_(gap) and 3′ tktprimers (SEQ ID NOs 1 and 11). Transaldolase and transketolaseactivities were also measured for isolates after the 1st and 10thtransfers (as described in General Methods). All 4 integrants hadsimilar levels of both TAL and TKT activities after 100 generations onthe non-selective medium, suggesting these integrants were geneticallystable.

Construction of pMODP_(gap)xylABCm for Transposition

The next step was to further integrate the P_(gap)xylAB loxP::Cm operoninto the ZW1::P_(gap)taltkt integrants (T2C, T3C, T4C and T5C). Theintegrative plasmid pMODP_(gap)xylABCm (FIG. 5) was constructed based onthe plasmid pMODP_(gap)taltktCm (FIG. 4). The P_(gap)taltkt DNA fragmentwas removed by SacI/SfiI digestion. An adaptor fragment containing SacI,NotI, and SfiI restriction sites was introduced by ligation. A NotIfragment of P_(gap)xylAB, that was isolated from pZB4 (U.S. Pat. No.5,514,583), was then cloned in the NotI site of the adaptor. Xyloseisomerase (XI) is encoded by xylA and xylulokinase (XK) is encoded byxylB. The complete nucleotide sequence for the pMODP_(gap)xylABCmplasmid is given as SEQ ID NO: 12.

Transposition and Transformation of pMODP_(gap)xylABCm in T2C, T3C, T4Cand T5C

Using a similar approach to the integration of P_(gap)taltktCm, T2C,T3C, T4C and T5C were transformed/transposed with pMODP_(gap)xylABCm(described above) treated with transposase. Six integrants (T3CCmX1,T3CCmX2, T3CCmX3, T4CCmX1, T5CCmX1, T5CCmX2) were obtained in 2transformation/transposition experiments following Cm selection. Allwere confirmed for the presence of xylAB by PCR using two sets ofprimers: SEQ ID NOs:13, and 14, and SEQ ID NOs: 15 and 16 except forT2CcmX1 and T2CcmX6 from which no PCR fragment was detected using theprimers SEQ ID NOs:13 and 14

The integrants, including the 2 PCR negative lines, were assayed for XI,XK, TAL and TKT activities (General Methods). The results shown in FIGS.6 and 7 indicated that the six xylAB integrants T3CCmX1, T3CCmX2,T3CCmX3, T4CCmX1, T5CCmX1, and T5CCmX2 all had XI, XK, TAL and TKTactivities. XI and XK activities were newly acquired as compared to thenegative parental controls (FIG. 6). TAL and TKT activities weremaintained as in the parental controls. All results indicated that theproteins were made and functional. Enzyme activity levels varied, withTI and XK activities similar to those of ZW1 integrantstransformed/transposed with the same plasmid. The levels of activitiesof XI, XK, TAL and TKT were lower than those in strain 8b.

The integration of the xylAB operon was confirmed by Southernhybridization. Both genomic and plasmid DNA of the 6 lines were digestedwith SphI and hybridized to a digoxenin labeled xylB probe. A commonband of about 3 kb, which is generated from an SphI site in xylB andanother SphI site in the adjacent cloning sites on the PMOD vector, waspresent in all genomic DNA samples, and in addition, higher molecularweight hybridizing bands in the genomic DNA samples indicated that therewere four sites of integration for the P_(gap)xylAB operon in thechromosome. T3CCmX1 and T3CCmX2 appear to have the same integrationsite, T3CCmX3 and T4CCmX1 may have the same integration site, andT5CCmX1 and T5CCmX2 each have a separate integration site. Digestion ofthe same DNA with PstI followed by Southern hybridization with the tktprobe demonstrated that each integrant had the same hybridizationpattern as its respective parental strain.

Adaptation of the ZW1::P_(gap)taltktP_(gap)xylAB Cm Integrants on XyloseMedia

Despite the presence of all four enzymatic activities for xyloseutilization, previous observations (US Patent Application 20030162271)indicated that the integrants may not grow on xylose immediately. Growthon xylose may occur after prolonged incubation on xylose medium (eitherin test tubes or on plates), a process called adaptation.

The strains were adapted as follows. ZW1::P_(gap)taltktP_(gap)xylABCmintegrant strains were inoculated into test tubes and plates containingRMX (containing 10 g/l yeast extract, 2 g/l KH₂PO₄, 20 g/l or 2% (w/v)xylose as well as RMGX (RM with 0.025% (w/v) glucose, 4% (w/v) xylose).The low level of glucose was used to support initial growth to increasethe chance of mutation during adaptation. One of at least five attemptsat adaptation on xylose in both cultures and plates was successful.After 10 days of anaerobic incubation at 30° C., 17 and 19 colonies werevisible on MMGX plated with T3CCmX1 and T3CCmX2 cells, respectively. Thecolonies were small and looked unhealthy (transparent) on the plates.Twelve colonies (four from T3CCmX1 plating: T3CCmX11, T3CCmX12, T3CCmX13and T3CCmX110; eight from T3CCmX2 plating: T3CCmX24, T3CCmX25, T3CCmX26,T3CCmX27, T3CCmX28, T3CCmX29, T3CCmX211 and T3CCmX212) were inoculatedin RMGCm120 and transferred into 3 ml RMX for further adaptation toobtain lines that were able to grow faster on xylose.

Adaptation of integrants in test tubes containing 3 ml RMX was conductedat 30° C. OD₆₀₀ was constantly monitored in a Spectronic 601spectrophotometer. When the growth reached mid-log phase, the cultureswere transferred into fresh tubes of RMX. This process was continued for7 transfers. The growth rates and final ODs (non-linear readings) wereimproved over the transfers.

At the 6^(th) transfer, the cultures were streaked out on RMX plates toisolate single colonies. Three integrants grew faster than others on RMXstreaked plates: T3CCmX13, T3CCmX26 and T3CCmX27, which are referred toas X13, X26 and X27 in the tables and discussion below. To screen forthe best xylose growers, four large (L1-4) and four small (S1-4)colonies each for TX13, X26 and X27 were selected and grown in RMX testtubes so that growth, sugar utilization, and ethanol production could bemonitored. Colonies were grown overnight at 30° C. followed byinoculation of OD₆₀₀=0.05 into 3 ml of RMX in test tubes in duplicates.X27 grew more slowly in RMG than the other cultures and was inoculatedagain 6.5 hrs later. After 69 hrs (62.5 hrs for X27), samples were takenfor HPLC analysis (General Methods). FIG. 8 charts the average ethanolyield (% of theoretical yield) and xylose utilization (%) for culturesat 69 hours (62.5 hr for all X27 cultures). There was no significantdifference between the large and small colonies. Although theperformance of X27 was better as compared to X26 on xylose, it showedslower growth on glucose. Therefore, the top performers, large coloniesof X13 (X13L3) and X26 (X26L1), were chosen for further evaluation inpH-controlled fermentations. The fermentations were conducted in RMG(6%glucose), RMX(6% xylose) and RMGX(8%:4%; glucose:xylose) at 37° C. forstrains X13L3 and X26L1, as well as the control strain 8b. Fermentationof glucose by X13L3 and X26L1 grown in RMG(6%) and RMGX(8%:4%) proceededrather quickly. The fermentation of xylose in the RMGX(8%:4%) was slowerfor both X13L3 and X26L1 as compared to that of strain 8b. In addition,growth on RMX(6%) at 37° C. occurred after a long lag for both X13L3 andX26L1. Several isolates, X13b, X13c and X13FL, were recovered fromRMX(6%) fermentations. These isolates along with the original strainsX13a (an isolate of X13L3) and X26 were subjected to Cre treatment, asdescribed previously in this Example, to remove the Cm^(r) marker fromZW1::P_(gap)taltktP_(gap)xylABCm strains. The resulting Cre treated,Cm^(r)-free integrants were named: X13aC, X13bC, X13cC, X13FLC and X26C.

Adaptation of Integrants in Xylose Medium by Serial Transfers in RMX(5%)at 37° C.

As described earlier, adaptation of the initialZW1::P_(gap)taltktP_(gap)xylABCm strains on RMX at 30° C. greatlyimproved the growth of strains in these conditions. However, the adaptedstrains suffered a long lag during growth and fermentation in RMX(6%) at37° C. To further improve the integrants for xylose fermentation atpreferred process conditions including higher sugar concentration andtemperature, the evolutionary or adaptation process was continued inRMX(5%) at 37° C. Serial transfers were conducted and the best growerswere selected. Integrants used in this process included X13aC, X13bC,X13cC, X26C and X13FLC. These 5 strains were grown in RMX at 30° C. for6 transfers before being transferred to RMX(5%) at 37° C. for another 5to 16 transfers. During and after all the transfers cultures werestreaked on RMXplates and incubated at 37° C. to isolate singlecolonies. Large colonies were further streaked on RMX plates andincubated at 37° C. for 3 to 4 times to purify the colonies. Final largecolonies were selected for growth testing in RMX(5%) at 37° C.

Evaluation of Strains from Adaptation in RMX(5%) Medium at 37° C.

Eighteen colonies isolated after adaptation with serial transfers weretested in RMX(5%) test tubes at 37° C. initially. Twelve strains wereselected for a 2nd test tube evaluation. Strain 8b was included in allthe evaluations for comparison. The 18 colonies were grown up in RMG at37° C. overnight, centrifuged and the cells were inoculated into 4 ml ofRMX(5%) at 37° C., statically in test tubes for the 1^(st) evaluation.Based on the growth (OD₆₀₀, non-linear) and end point HPLC results (lowresidual xylose and high ethanol), 12 strains were selected for the2^(nd) evaluation.

One of the purposes of the 2^(nd) evaluation was to test the stabilityof improved growth on xylose and xylose utilization capability of thestrains. All 12 strains were subjected to a stability study to seewhether the adapted strains were stable after being exposed to anon-selective medium in which they were serially transferred in at 37°C. for 50 generations. Cultures before and after RMG(5%) transfers wereinoculated in RMX(5%) test tubes and grown at 37° C. for evaluation. Thenon-linear ODs were monitored by direct reading of test tubes in aSpectronic 601 spectrophotometer. The ODs at the 70^(th) hour of growthin RMX(5%) before and after 50 generations of growth in RMG are plottedin FIG. 9. The results indicated that most strains were stable after 50generations in RMG at 37° C. The endpoint (at stationary phase)supernatants were also analyzed by HPLC for xylose and ethanolconcentrations. The low residual xylose and high ethanol concentrationsin these cultures supported the fact that the strain grew and fermentedxylose well.

Based on the results from the above test tube evaluation (low residualxylose, high ethanol concentration and higher OD) and a subsequentmicrotiter plate growth screening with high concentrations of glucoseand/or xylose (up to 20%) and mixtures of glucose and xylose withacetate to select better growers in high sugars and in the presence ofacetate, we decided to focus on strain #26, designated as ZW658, whichexhibited the best overall performance

Example 2 Fermentation Evaluation of Top Improved Xylose-UtilizationStrains at 37° C.

The following example illustrates the fermentation performance of theimproved xylose-utilizing Zymomonas strain ZW658 under fermentationconditions that mimic the sugar concentrations and the acetic acid levelexpected in a biomass hydrolysate. Strain ZW658 was inoculated intofermentors containing RM medium supplemented with 10% glucose (RMG10%),8% xylose (RMX8%), 10% glucose+8% xylose (RMGX10%8%) and 10% glucose+8%xylose+0.6% acetic acid (RMGXAc10%8%0.6%), respectively. Allfermentations were conducted in Sixfors with 300 ml media at 150 rpm,pH5.5 and 37° C. Nitrogen was purged through the media in the fermentorsovernight and stopped right before inoculation. No nitrogen was purgedduring the fermentation. Inocula for the fermentation were prepared withRMGX(10%,4%) at 37° C. in shake flasks (150 rpm) after reviving of theworking stocks in RMG5%. Strain 8b was used as a control under the sameconditions. As shown in FIG. 10, ZW658 grew more slowly on RMG10% ascompared to 8b (A and B), and grew at a similar rate to 8b on RMX8% (Cand D). Despite the slower growth rate, FIG. 10 shows that the ethanolyield of ZW658 (93%) was similar to that of 8b at the end offermentation in glucose medium. In RMX8% medium, the ethanol yield washigher for ZW658 (0.46 g ethanol/g sugar) as compared to 8b (0.44 gethanol/g sugar). ZW658 produced about 4 g/1 more ethanol as compared to8b in RMX8%. Interestingly, ZW658 did not produce any xylitol while 8bproduced a low level of xylitol (0.7 g/l) at the end of the fermentationin RMX8%. Data shown in FIG. 11 shows that ZW658 performed better ascompared to 8b in fermenting 10% glucose+8% xylose with (C, D) orwithout (A, B) acetate, indicated by more glucose and xyloseconsumption, less xylitol production, and more ethanol production. Mostof the glucose was used and substantial residual xylose remained at theend of the fermentation for both strains in RMG10% X8%, at 37° C. andpH5.5, although ZW658 used about 8 g/1 more xylose than 8b. Xylitolproduction (4.9 g/l) in ZW658 in RMG10% X8% at 37° C. and pH5.5 at theend of the fermentation was significant lower than that of 8b (8.2 g/l).In the presence of acetate (6 g/l), the cell growth of both strains wasreduced significantly resulting in poor fermentation performance of bothglucose and xylose, although ZW658 showed slightly better fermentationperformance in terms of more glucose and xylose consumption, lessxylitol production and more ethanol production. Unlike in the RMX8%,both strains produced the by-product xylitol in RMG10% X8% with orwithout acetate, although less xylitol was produced by ZW658 as comparedto 8b. The fermentation performance of the two strains is summarized inTable 1. Overall, ZW658 performed better than 8b in pure sugarfermentations. As described in Example 1, ZW658 is free of antibioticselection markers, which is a valuable property for fermentationorganisms in commercial applications.

TABLE 1 Summary of fermentation performance of ZW658 and 8b for ethanolproduction. Ethanol Yield Vol. Prod. Ethanol CPI g ethanol/g sugar g/l/hg/l g/g 8b (Glu) 0.47 5.15 52 21 ZW658 (Glu) 0.48 4.13 52 15 8b (Xyl)0.44 1.66 37 24 ZW658 (Xyl) 0.46 1.83 41 23 8b (Glu Xyl) 0.43 1.80 58 52ZW658 (Glu Xyl) 0.45 2.03 65 35 8b (Glu Xyl Ac) 0.46 0.67 48 136 ZW658(Glu Xyl Ac) 0.47 1.04 50 90 CPI is Cell Productivity Index: g ethanol/gdry cell weight

Example 3 Preparation of a Suicide Construct forInsertional-Inactivation of the Glucose-Fructose Oxidoreductase (GFOR)Gene in ZW1 and ZW658

The suicide construct used to knockout the gene encodingglucose-fructose oxidoreductase in ZW1 and ZW658 (“GFORSp-9WW”) wasderived from another suicide construct (“pLDHSp-9WW”) that was usedpreviously to insertionally-inactivate the gene for D-lactatedehydrogenase in Z. mobilis using host-mediated, double-crossover,homologous recombination and spectinomycin resistance as a selectablemarker. pLDHSp-9WW was also derived from a number of other constructsthat were previously generated. The initial precursor for all of theseconstructs was the plasmid vector pNEB193 (NEB #N3051S; New EnglandBiolabs). This plasmid was chosen because it can replicate in E. colibut it cannot replicate in Z. mobilis. All of the steps andintermediates that were involved in generating the GFOR knockoutconstruct are described below in chronological order starting withplasmid pNEB193.

Construction of pLDH193

pNEB193 was double-digested with SbfI and AscI for insertion of the DNAfragment that is described below. Both restriction sites are unique andare located in the multi-cloning region of the plasmid. TheSbfI/AscI-linearized pNEB193 plasmid DNA fragment was purified usingQiagen's QIAQuick Purification Kit (catalog #28104) according to themanufacturer's protocol. The DNA fragment that was cloned into pNEB193was a 2268 bp fragment that was PCR-amplified from Z. mobilis genomicDNA, that was isolated from strain ZW1 using Qiagen's Blood & CellCulture Maxi Kit (catalog #13362). The synthetic oligonucleotides thatwere used for PCR-amplification of this fragment were Primers 1 and 2:

Primer 1 (SEQ ID NO:17) CTACTCATTTcctgcaggTGGTAACTCATTGCGCGCTC Primer 2(SEQ ID NO:18) CATCTTACTggcgcgccAAAAATCTGCGGCTGACATACThe underlined bases of Primer 1 (forward primer) hybridize tonucleotides 1262739-1262720 of GenBank accession number AE008692 at the3′ end of the open reading frame that codes for phosphoglyceromutase(pgm), while the lower case letters correspond to an SbfI site that wasadded to the 5′ end of the primer. The underlined bases of Primer 2(reverse primer) hybridize to nucleotides 1260490-1260472 of GenBankaccession number AE008692, which is just upstream from the open readingframe that codes for alcohol dehydrogenase I (adhI), while the lowercase letters correspond to an AscI site that was added to the 5′ end ofthe primer. The 2268 bp DNA fragment that was the target forPCR-amplification therefore consists of the following elements startingfrom the SbfI site and ending at the AscI site: (a) the 3′ end of thepgm gene, (b) the entire Idh gene that codes for D-lactatedehydrogenase, and (c) a 5′ non-translated region of the adhI gene. ThePCR product was cut with SbfI and AscI, and the resulting DNA fragmentwas ligated into the SbfI/AscI-linearized pNEB193 vector that wasdescribed above. The ligation reaction mixture was used to transform E.Coli JM110 and the transformed cells were plated on LB medium thatcontained ampicillin (100 μg/ml). Ampicillin-resistant tranformants thatcontained plasmids with the correct size insert were initiallyidentified by PCR using resuspended colonies (“colony PCR”) and Primers1 and 2. Subsequent confirmation of positive clones came fromrestriction digestion analysis of plasmid DNA with SbfI and AscI, andDNA sequence analysis of the 2268 bp fragment that was generated bycolony PCR with the ampicillin-resistant transformants. The plasmid thatwas selected for further manipulation is referred to below as pLDH193.Construction of pLDHTc139#7

Plasmid pLDH193 has a unique NcoI site that is located near the middleof the Idh open reading frame. This site was used to insert a DNAfragment that confers resistance to tetracycline. The tetracyclineresistance cassette (Tc^(r)-cassette) that was used for thismanipulation was generated by PCR using plasmid pACYC184 (GenBankaccession number X06403) as a DNA template and Primers 3 and 4 as PCRprimers.

Primer 3 (SEQ ID NO:19) ACTCATTTccatggCGATCGCACTATgcggccgcAATGTAGCACCTGAAG TCAGCC Primer 4 (SEQ ID NO:20)ATCTCACTccatggCCGGCCAACTAttaatt aaGAATTGATTGGCTCCAA TTCTTGThe bold underlined bases of Primer 3 (forward primer) hybridize justupstream from the promoter for the tetracycline resistance gene. Primer3 also has three restriction sites (NcoI, AsiSI, and NotI) that wereadded to its 5′ end. The NcoI site is in lower case letters. The AsiSIsite is underlined with a thin line. The Not I site is in italicizedlower case letters. The bold underlined bases of Primer 4 (reverseprimer) hybridize just downstream from the stop codon for thetetracycline resistance gene, and this primer also has three restrictionsites (NcoI, FseI, and PacI) that were added to its 5′ end. Similar tothe labeling above, the NcoI site is in lower case letters, the FseIsite is underlined with a thin line, and the PacI site is in italicizedlower case letters. The 1448 bp Tc^(r)-cassette that was generated withPrimers 3 and 4 was cut with NcoI and purified by preparative agarosegel electrophoresis. The resulting DNA fragment was then ligated intothe unique NcoI site that is present in the Idh open reading frame ofplasmid, pLDH193. To minimize the possibility of re-circularization ofthe vector without an insert, the NcoI-digested pNEB193 wasdephosphorylated with calf intestinal alkaline phosphatase prior toligation. The ligation reaction mixture was introduced into Escherichiacoli JM110 and the transformed cells were plated on LB medium thatcontained 20 μg/ml of tetracycline. Tetracycline-resistant tranformantsthat contained plasmids with the correct insert were identified byrestriction digest analysis with NcoI, AsiSI, NotI, FseI, and PacI, andthe orientation of the Tc^(r)-cassette was confirmed by PCR analysisusing appropriate primers. A circle diagram of the plasmid that wasselected for further manipulation (pLDHTc139#7) is shown in FIG. 12. Inexperiments not described here, this suicide construct was used toinsertionally-inactivate (i.e. “disrupt” or “knockout”) the D-lactatedehydrogenase gene in ZW1 using host-mediated, double-crossover,homologous recombination and growth on tetracycline as the selection.Construction of pLDHTc139#7-9WW

Having demonstrated that pLDHTc139#7 could be used to “knockout” theD-lactate dehydrogenase gene in ZW1, the next step was to modify thisconstruct so that it would be possible to remove the selectable markerfrom the chromosome after gene disruption, using Cre recombinase. Toaccomplish this goal, two wild type loxP sites (Lee and Saito, 1998)were added to pLDHTc139#7 taking advantage of the four uniquerestriction sites that flank the Tc^(r)-cassette, namely, AsiSI and NotIat the 5′ end and PacI and FseI at the 3′ end. The first loxP site wasinserted between the AsiSI and NotI sites of plasmid pLDHTc139#7 aftercutting the construct with both enzymes and purifying the resultinglarge DNA fragment. The loxP site that was inserted into this locationwas generated from two synthetic oligonucleotides 5 and 6 (SEQ ID NOs:21and 22) that were both phosphorylated at their 5′ end.

Oligonucleotide 5; (SEQ ID NO:21)cgcATAACTTCGTATAATGTATGCTATACGAAGTTATgc Oligonucleotide 6; (SEQ IDNO:22) ggccgcATAACTTCGTATAGCATACATTATACGAAGTTATgcgatThese oligonucleotides are complimentary to each other, and whenannealed together form a full-length double-stranded wild type loxP sitethat has single-stranded overhangs at both ends, which allow the DNAfragment to be ligated between the AsiSI and NotI sites of pLDHTc139#7.The upper case letters in the oligonucleotides correspond to thefull-length wild type loxP site, while the lower case letters indicatethe nucleotides that were used to ligate the double-stranded DNAfragment into the AsiSI and NotI sites of pLDHTc139#7.

The ligation reaction mixture was used to transform E. coli DH10B andthe transformed cells were plated on LB medium that contained 20 μg/mlof tetracycline. Tetracycline-resistant tranformants that containedplasmids with the loxP site correctly inserted into the AsiSI and NotIsites of pLDHTc139#7 were identified by restriction digest analysis,colony PCR, and DNA sequence analysis of the relevant regions. Theplasmid that was selected for further manipulation is referred to belowas pLDHTc139#7-9W.

Next, a second wild type loxP site was inserted between the PacI andFseI sites at the other end of the Tc^(r)-cassette in pLDHTc139#7-9W,after cutting the plasmid with both enzymes and purifying the resultinglarge vector fragment. The loxP site that was inserted into thislocation was also generated with two synthetic oligonucleotides 7 and 8(SEQ ID NOs: 23 and 24) that were both phosphorylated at their 5′ end.

Oligonucleotide 7: (SEQ ID NO:23)taaATAACTTCGTATAATGTATGCTATACGAAGTTATggccgg Oligonucleotide 8: (SEQ IDNO:24) ccATAACTTCGTATAGCATACATTATACGAAGTTATttaatOligonucleotides 7 and 8 are complimentary to each other, and whenhybridized form a full-length, double-stranded wild type loxP site thathas single-stranded overhangs at both ends that allow the DNA fragmentto be ligated between the PacI and FseI sites of pLDHTc139#7-9W. Theupper case letters in the oligonucleotides correspond to the full-lengthloxP site, and the lower case letters indicate the nucleotides that wereused to ligate the double-stranded DNA fragment into the PacI and FseIsites of pLDHTc139#7-9W.

The ligation reaction mixture was used to transform E. coli DH10B andthe transformed cells were plated on LB medium that contained 20 μg/mlof tetracycline. Tetracycline-resistant tranformants that containedplasmids with the wild type loxP site correctly inserted into the PacIand FseI sites of pLDHTc139#7-9W were identified by restriction digestanalysis, colony PCR, and DNA sequence analysis of the relevant regions.The plasmid that was selected for further manipulation is referred tobelow as pLDHTc139#7-9WW, and a circle diagram of this construct isshown in FIG. 12.

Construction of pLDHSp-9WW

pLDHSp-9WW is identical to pLDHTc139#7-9WW, but thetetracycline-resistance cassette in the latter construct was replacedwith a DNA fragment that confers resistance to spectinomycin (i.e. aSpec^(r)-cassette). The latter was generated by PCR using plasmidpHP15578 (Cahoon et al, 2003) as a template and Primers 9 and 10.pHP15578 contains the complete nucleotide sequence for theSpec^(r)-cassette and its promoter, which is based on the publishedsequence of the Tranposon Tn7 aadA gene (GenBank accession numberX03403) that codes for 3′ (9)-O-nucleotidyltransferase.

Primer 9 ATAAAAgcggccgcAGCACAGGATGA (SEQ ID NO:25) Primer 10GGCGttaattaaGGCAGGTCAGCAAG (SEQ ID NO:26)

The underlined bases of Primer 9 (forward primer) hybridize justupstream from the promotor for the Spec^(r)-cassette (to nts 6-17 ofGenBank accession number X03043), while the lower case letterscorrespond to a NotI site that was added to the 5′ end of the primer.The underlined bases of Primer 10 (reverse primer) hybridize about 130bases downstream from the stop codon for the Spec^(r)-cassette (to nts1006-1019 of GenBank accession number X03043), while the lower caseletters correspond to a PacI site that was added to the 5′ end of theprimer. The 1040 bp PCR-generated Spec^(r)-cassette was double-digestedwith NotI and PacI, and the resulting DNA fragment was purified byagarose gel electrophoresis. Plasmid pLDHTc139#7-9WW was also cut withthe same two

restriction enzymes to remove the Tc^(r)-cassette, and the resultinglarge vector fragment was purified by agarose gel electrophoresis. Thetwo DNA fragments of interest were then ligated together, and thetransformation reaction mixture was introduced into E. coli DH10B usingelectroporation. Transformants were plated on LB medium that containedspectinomycin (200 μg/ml) and grown at 37° C. Spectinomycin-resistanttranformants that contained plasmids with the correct size insert wereidentified by restriction digest analysis with NotI and PacI, and theplasmid that was selected for further manipulation is referred to belowas pLDHSp-9WW; a circle diagram of this construct is shown in FIG. 12.In experiments not described here, pLDHSp-9WW was used to knockout thegene for D-lactate dehydrogenase in ZW1 using resistance tospectinomycin as the selectable marker. Gene inactivation with thesuicide construct occurred via host-mediated, double-crossover,homologous recombination, (WO 01/83784 A2), which resulted in theinsertion of the selectable marker (the Spec^(r)-cassette) that isflanked by two wild type loxP sites in the middle of the Idh openreading frame. The double-crossover event was targeted to the Idh geneby two DNA fragments that flank the Spec^(r)-cassette in pLDHSp-9WW. Oneof these fragments (referred to below as 5′ Idh flanking DNA) is justupstream from the Spec^(r)-cassette and is located between the SbfI andAsiSI sites. The nucleotide sequence of this ˜1100 bp DNA fragment isidentical to the ZW1 chromosomal DNA that codes for the 3′ end of thepgm gene and about the first half of the Idh open reading frame. Theother DNA fragment (referred to below as the 3′ Idh flanking DNA) islocated at the opposite end the Spec^(r)-cassette between the FseI andAscI sites. The nucleotide sequence of the 3′ Idh flanking DNA (which isalso ˜1100 bp) is identical to the chromosomal DNA that codes for theother half of the Idh gene and part of the 5′ non-translated region ofthe adhI gene. A double-crossover event occurs when the 5′ and 3′ Idhflanking DNA fragments both interact with their chromosomal counterpartsand undergo homologous recombination. This phenomenon, which isessentially irreversible and entirely mediated by the host's enzymaticmachinery, inactivates the chromosomal Idh gene by inserting theSpec^(r)-cassette in the middle of the open reading frame. Since theconstruct cannot replicate in Z. mobilis, making it a suicide construct,the only way to generate stable spectinomycin-resistant colonies withpLDHSp-9WW (apart from spontaneous drug resistant mutants that occur ata very low frequency) is a double-crossover event through homologousrecombination. It is important to note that the Spec^(r)-cassette thatgets inserted into the chromosome by the double-crossover event is stillsandwiched between the two wild type loxP sites that were present in thesuicide construct. Because of this arrangement it is easy to remove theselectable marker from the D-lactate dehydrogenase gene withoutreactivating it by using the Cre Expression vector that is described inExample 10.Construction of pGFORSp-9WW

pLDHSp-9WW was converted to a suicide construct for gene inactivation ofZ. mobilis glucose-fructose oxidoreductase (GFOR) in a 2-step procedureas described below. The first step was to remove the 3′ Idh flanking DNAand replace it with an analogous DNA fragment that would target theplasmid construct to the chromosomal gene that codes for GFOR. Thelatter DNA fragment (referred to below as 3′ GFOR flanking DNA) wasgenerated by PCR using ZW1 genomic DNA as a template and Primers 11 and12 as PCR primers.

Primer 11 (SEQ ID NO:27) CTACTCATggccggccTCAGAACGATCCTGCACAGC Primer 12(SEQ ID NO:28) CATCTTACTggcgcgccGGACGAGGTTCATCATCAGGThe underlined bases of Primer 11 (forward primer) hybridize tonucleotides 684324-684305 of GenBank accession number AE008692 which areapproximately in the middle of the GFOR open reading frame, while thelower case letters correspond to an FseI site that was added to the 5′end of the primer. The underlined bases of Primer 12 (reverse primer)hybridize to nucleotides 683124-683143 of GenBank accession numberAE008692 which is ˜625 bp downstream from the GFOR stop codon, while thelower case letters correspond to an AscI site that was added to the 5′end of the primer. The 1234 bp PCR fragment was cut with FseI and AscI.pLDHSp-9WW was also cut with the same restriction enzymes to remove the3′ Idh flanking DNA, and the large vector fragment resulting from thismanipulation was purified by agarose gel electrophoresis. ThePCR-generated 3′ GFOR flanking DNA was then ligated between the FseI andAscI sites of the gel purified large vector fragment described above,and an aliquot of the ligation reaction mixture was electroporated intoE. coli DH10B. The transformed cells were plated on LB medium thatcontained 200 μg/ml of spectinomycin and the plates were incubated at37° C. Spectinomycin-resistant transformants that contained plasmidswith the correct insert were identified by colony PCR and restrictiondigestion analysis with FseI and AscI, and the plasmid that was selectedfor further manipulation is referred to below as pLDH/GFORSp-9WW.

The next step was to remove the 5′ Idh flanking DNA from pLDH/GFORSp-9WWand replace it with 5′ GFOR flanking DNA, so a double-crossover eventcould occur at the chromosomal targets that were selected for disruptionof the GFOR open reading frame. The 5′ GFOR flanking DNA fragment wasgenerated by PCR using ZW1 genomic DNA as a template and Primers 13 and14 as PCR Primers.

Primer 13: CTACTCATatgcatGTCCAGAAAAGACAGCATTCC (SEQ ID NO:29) Primer 14:CATCTTACTgcgatcgcTGCACGGTTCATTGGAT (SEQ ID NO:30)The underlined bases of Primer 13 (forward primer) hybridize tonucleotides 685584-685564 of GenBank accession number AE008692 which areapproximately 520 bp upstream from the GFOR start codon, while the lowercase letters correspond to an NsiI site that was added to the 5′ end ofthe primer. The underlined bases of Primer 14 (reverse primer) hybridizeto nucleotides 684396-684415 of GenBank accession number AE008692 whichare close to the middle of the GFOR open reading frame and just upstreamfrom the binding site for Primer 11, while the lower case letterscorrespond to an AsiSI site that was added to the 5′ end of the primer.The 1217 bp PCR product was cut with NsiI and AsiSI, and pLDH/GFORSp-9WWwas double-digested with SbfI and AsiSI to remove the 5′ Idh flankingDNA; the large vector fragment resulting from the latter manipulationwas purified by agarose gel electrophoresis. The PCR-generated 5′ GFORflanking DNA was then ligated into the SbfI and AsiSI sites of the gelpurified large vector fragment described above, and an aliquot of theligation reaction mixture was electroporated into E. coli SCS110 (whichis dcm⁻¹ and dam⁻) to obtain non-methylated plasmid DNA for subsequenttransformation of ZW1 and ZW658, which is described in detail below inExamples 5 and 7. Note that the use of non-methylated plasmid DNA fortransformation of Z. mobilis stains that are derived from ZM4 iscritical for success, since methylated plasmid DNA that is isolated fromwild type E. coli strains, like DH10B, is readily destroyed by thehost's restriction/modification system (described in U.S. Pat. No.6,566,107 B1). Note further that NsiI and SbfI have compatible stickyends, but both sites are destroyed when they are ligated together.Transformants were plated on LB medium that contained 100 μg/ml ofspectinomycin and the plates were incubated at 37° C.Spectinomycin-resistant transformants that contained plasmids with thecorrect insert were identified by colony PCR and restriction digestionanalysis. This resulting suicide construct that was used to knockout theGFOR gene in ZW1 and ZW658 is referred to below as pGFORSp-9WW. A circlediagram of this plasmid is shown in FIG. 12, and its complete nucleotidesequence is disclosed in SEQ ID NO:31. It is important to note that adouble-crossover event between this suicide construct and the Z. mobilischromosome results in the insertion of a Spec^(r)-cassette that isflanked by two wild type loxP sites, analogous to the situationdescribed above for pLDH-Spec-9WW.

Example 4 Generation of an E. coli Xylose Isomerase Expression Vectorfor Z. mobilis

A plasmid construct for expression of E. Coli xylose isomerase in Z.mobilis (pZB188/Kan-XylA) was generated as described below using an E.coli/Z. mobilis shuttle vector (pZB188) as starting material (FIG. 13).Steps involved in the construction of pZB188 are disclosed in U.S. Pat.No. 5,514,583. Briefly, this 7008 bp plasmid is able to replicate in E.coli and Z. mobilis because it has two different origins of replication,one for each bacterial species. pZB188 also contains a DNA fragment thatconfers resistance to tetracycline (i.e. a Tc^(r)-cassette). The firststep in the construction of pZB188/Kan-XylA, was to remove theTc^(r)-cassette from pZB188 and replace it with a DNA fragment thatconfers resistance to kanamycin (i.e. Kan^(r) cassette). To excise theTc^(r)-cassette from pZB188, the plasmid was cut with XbaI and BssHIIand the resulting large vector fragment was purified by agarose gelelectrophoresis. The Kan^(r)-cassette was generated by PCR using plasmidpET-24a (Novagen) as a template and Primers 15 and 16 forPCR-amplification. pET-24a contains the complete open reading frame forthe Kan^(r) gene and its associated promoter.

Primer 15: GCtctagaGCAGCAGATTACGCGC (SEQ ID NO:32) Primer 16:ACATTGgcgcgcTTAGAAAAACTCATC (SEQ ID NO:33)The underlined bases of Primer 15 (forward primer) hybridize about 160bp upstream from the start codon for the Kan^(r) gene in pET-24a, whilethe lower case letters correspond to an XbaI site that was added to the5′ end of the primer. The underlined bases of Primer 16 (reverse primer)hybridize at the other end of the open reading frame for the Kan^(r)gene and include the termination codon, while the lower case letterscorrespond to a BssHII site that was added to the 5′ end of the primer.The 991 bp PCR-generated Kan^(r)-cassette was cut with XbaI and BsshII,and purified by agarose gel electrophoresis.

The resulting DNA fragment was then inserted between the XbaI and BssHIIsites of the pZB188 DNA fragment described above in a standard ligationreaction. The transformation reaction mixture was introduced into E.coli DH10B using electroporation and the cells were plated on LB mediumthat contained kanamycin (50 μg/ml); growth was at 37° C. Plasmid DNAwas isolated from one of the kanamycin-resistant transformants, and theresulting construct is referred to below as pZB188/Kan; a circle diagramof this shuttle vector is shown in FIG. 13.

In the next step, an E. coli xylose isomerase expression cassette wasinserted between the NcoI and AcII sites of pZB188/Kan after cutting thelatter with both enzymes, and purifying the large vector fragment byagarose gel electrophoresis. The ˜2 Kbp DNA fragment that served as theE. coli xylose isomerase expression cassette was derived from plasmidpZB4 after cutting the latter construct with NcoI and ClaI, andpurifying the relevant DNA fragment by agarose gel electrophoresis.Plasmid pZB4 is described in detail in U.S. Pat. No. 5,514,583, and aschematic representation of the E. coli expression cassette P_(gap)XylA(SEQ ID NO:34) is shown in the boxed diagram of FIG. 13.

NcoI and ClaI sites were located at the 5′ and 3′ ends, respectively, ofthe E. coli xylose isomerase expression cassette. As described in U.S.Pat. No. 5,514,583, this fragment contains the strong, constitutive Z.mobilis glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter, whichis precisely fused to the complete open reading frame of the E. colixylA gene that codes for xylose isomerase. It also contains the smallstem-loop region that immediately follows the xylose isomerase stopcodon. The E. coli xylose isomerase expression cassette was insertedbetween the NcoI and AclI sites of pZB188/Kan in a standard ligationreaction. Note that ClaI and AclI generate compatible “sticky ends”, butboth sites are destroyed when they are ligated together. The ligationreaction mixture was then electroporated into E. coli SSC110 (dcm⁻¹,dam⁻) to obtain non-methylated plasmid DNA for subsequent transformationof Z. mobilis as described below in Example 6, and transformed cellswere plated on LB medium that contained kanamycin (50 μg/ml); growth wasat 37° C. Kanamycin-resistant tranformants that had a plasmid with acorrect size insert were identified by restriction digestion analysisand colony PCR. The plasmid that was used to express E. coli xyloseisomerase in Z. mobilis is referred to below as “pZB188/Kan-XylA”; acircle diagram of this construct is shown in FIG. 13.

Example 5 Generation of the ZW1 GFOR Knockout Mutant

To eliminate GFOR enzyme activity in ZW1 (the wild type strain thatZW658 was originally derived from) the suicide construct pGFORSp-9WW,which was described in detail in Example 3, was used. Thenon-replicating plasmid DNA was introduced into the bacterial host usingelectroporation, essentially as described in U.S. Pat. No. 5,514,583.Briefly, the 50-μl transformation reactions contained ˜10¹⁰ cells/ml in10% (v/v) glycerol and ˜0.9 μg of non-methylated plasmid DNA that wasisolated from E. coli SSC110 as described in Example 3. The controlreaction was treated identically, but did not receive any plasmid DNA.The settings for the electroporator were 16 kv/cm, 200Ω, and 25 μF, andthe gap width of the cuvette was 0.1 cm. After electroporation, thetransformation reactions were diluted with 1.0 ml of MMG media (50 g/Lglucose, 10 g/L yeast extract, 5 g/L of tryptone, 2.5 g/L of (NH₄)₂SO₄,0.2 g/L K₂HPO₄, and 1 mM MgSO₄) and the cells were allowed to recoverfor ˜5 hours at 30° C. The cells were then harvested by centrifugationat room temperature (13,000×g, 5 min) in sterile 1.5-ml microfuge tubesand the supernatants were carefully removed. Cell pellets wereresuspended in 150 μl of liquid MMG media, and 50- and 100-μl aliquotsof the cell suspension were plated on MMG medium that contained 1.5%agar and 200 μg/ml of spectinomycin. The plates were incubated in ananaerobic chamber at 30° C., and 50 to 150 colonies appeared on theexperimental plates after 2 to 3 days. No spectinomycin-resistantcolonies were on the control plates at this time, although a fewappeared after another 48-hr incubation period. Two of thespectinomycin-resistant colonies that resulted from transformation withthe GFOR knockout construct were selected for further manipulation asdescribed below.

Previous experiments with Z. mobilis and suicide constructs that aresimilar to pGFORSp-9WW have revealed that the initial interactionbetween the chromosome and the plasmid DNA is a single-crossover eventat one of the two targeted loci, and that single-crossover eventseventually give rise to double-crossover events. Transition to thedouble-crossover event normally occurs very rapidly after a few serialtransfers in liquid medium that contains the selective agent for thesuicide construct. To facilitate the double-crossover event for the twoselected ZW1 transformants that resulted from the GFOR knockoutconstruct, cells were inoculated into 10 ml of RM media (10 g/L of yeastextract and 2 g/L of KH₂PO₄) that contained 100 g/L of glucose and 200μg/ml of spectinomycin. Both cultures reached stationary phase after a24-hr incubation period at 30° C. Next, 10 μl-aliquots of the1^(st)-pass cultures were used to inoculate 10 ml of the same growthmedium, and both of these cultures also reached stationary phase after24 hrs at 30° C. Finally, 10-μl aliquots of the 2^(nd)-pass cultureswere inoculated into 10 ml of the same growth medium and growth wasallowed to proceed for another 24 hrs at 30° C. Following the lasttransfer in liquid medium, aliquots of the 3^(rd)-pass cultures werediluted and plated on MMG medium that contained spectinomycin (200μg/ml) to obtain single colonies, and the plates were incubated at 30°C. for 48 hr under anaerobic conditions.

Confirmation that the double-crossover event had indeed occurred wasobtained from colony PCR experiments using three different pairs ofprimers. The first pair of PCR primers can only generate a DNA fragmentof the correct size if the 5′ GFOR flanking DNA in the suicide constructhas undergone a single-crossover event with its chromosomal counterpart.Similarly, the second pair of PCR primers can only generate a DNAfragment of the correct size if the 3′ GFOR flanking DNA in the suicideconstruct has undergone a single-crossover event with its chromosomalcounterpart. Finally, the third pair of PCR primers can only generate aDNA fragment of the correct size if a double-crossover event hasoccurred and in addition rules out the possibility of a mixed populationof single- and double-crossover events. The two spectinomycin-resistantcolonies that were used for this analysis were derived from twodifferent primary transformants from the ZW1 electroporation reactionwith the suicide construct that were transferred three times in liquidmedium and plated to obtain single colonies as described above, and thecontrol for this experiment was the parent strain, ZW1. Since bothtransformants yielded positive results with the three different sets ofPCR primers, only one of them was selected for further analysis. Thisstrain (the ZW1 GFOR knockout mutant) is referred to below as ZW1-ΔGFOR.

Example 6 Glucose-Fructose Oxidoreductase can be a Major Contributor toXylitol Formation Under Physiological Conditions

The ZW1 GFOR knockout mutant (ZW1-ΔGFOR) was used to test the hypothesisthat xylitol formation in xylose-utilizing, recombinant strains of Z.mobilis is at least partially mediated by the periplasmic enzyme GFOR,or its larger molecular weight cytosolic precursor which is alsoenzymatically active (Loos et al., supra). As shown in Example 2 (FIG.11), xylitol is a major by-product of xylose-utilizing strains 8b andZW658, but is only formed when xylose is present in the growth medium.Although wild type strains of Z. mobilis, like CP4, have anNADPH-dependent aldose reductase that can directly reduce xylose toxylitol (Feldmann et al, supra), it is conceivable that GFOR could alsocontribute to xylitol formation in vivo when the growth medium containsxylose or a mixture of glucose and xylose as depicted in Diagrams II andIII. However, for either of these reactions to occur the enzyme wouldneed access to xylulose, since this compound is the obligatory electronacceptor for GFOR-mediated xylitol production as shown in in vitro GFORenzyme characterization assays (Zachariou and Scopes, supra). In Z.mobilis strains that are engineered for growth on xylose, the xylulosethat would be necessary for xylitol synthesis would be generated byxylose isomerase, which catalyzes the first step of xylose metabolism(FIG. 1) and is absent in wild type strains, like ZW1

To test the possibility that GFOR can generate xylitol when xylose andglucose are both present in the growth medium, the E. coli xyloseisomerase expression vector (pZB188/Kan-XylA) that was described inExample 4 was introduced into ZW1 and ZW1-ΔGFOR. The strategy was toprovide a route from xylose to xylulose in two strains that cannot growon either of these sugars and determine whether GFOR could generatexylitol. The electroporation procedure that was used for transformationwas essentially as described in Example 5, but after the recovery periodthe transformed cells were plated on MMG medium that contained 300 μg/mlof kanamycin. As controls for this experiment, ZW1 and ZW1-ΔGFOR werealso transformed with pZB188/Kan (FIG. 13), which is identical topZB188/Kan-XylA but lacks the E. coli xylose isomerase expressioncassette. Kanamycin-resistant colonies harboring the pZB188/Kan-XylA orthe control plasmid were identified by colony PCR, and a representativecolony from each transformation reaction was randomly selected for theexperiment that is shown in FIG. 14. These four plasmid-bearing strainsare referred to below as ZW1 (pZB188/Kan), ZW1 (pZB188/Kan-XylA),ZW1-ΔGFOR(pZB188/Kan) and ZW1-ΔGFOR (pZB188/Kan-XylA).

Overnight cultures were grown in 15-ml capped test tubes at 30° C. in 5ml of 60 g/L glucose, 10 g/L yeast extract, 10 g/L KH₂PO₄, 2 g/L(NH₄)₂SO₄, 1 g/L MgSO₂(7H₂O) and 300 μg/ml of kanamycin. Aliquots ofthese overnight cultures were then used to inoculate 20 ml cultures (in50-ml capped test tubes) that contained the same growth medium, with orwithout 20 g/L of xylose. Growth was at 30° C. with gentle agitation,and initial OD₆₀₀ values were ˜0.1. After 0, 24, 48, and 120 hours ofgrowth, 1.0-ml aliquots of the cultures were removed for HPLC analysisusing an HP 1100 equipped with a refractive index detector(Hewlett-Packard, Palo Alto, Calif.) to determine the concentrations ofxylose, xylulose and xylitol that were present in the fermentationbroth. Prior to HPLC analysis, cells were removed by centrifugation andthe supernatant was filtered through a 0.22 μm cellulose acetate Spin-Xcentrifuge tube filter (Costar, catalog number 8160) to remove smallparticles. Compounds were separated on an Aminex HPX-87H column(Bio-Rad) that was run at 55° C. under isocratic conditions using a flowrate of 0.6 ml/min and 0.01 NH₂SO₄ as the mobile phase. Authenticstandards of known concentration were used to quantify the peaks ofinterest and all results are expressed in g/L.

The results show that when ZW1 (pZB188/Kan), the control strain with the“empty” vector, was grown in the presence of glucose and xylose, only asmall amount of xylitol accumulated in the growth medium after a 120-hrincubation period (FIG. 14A). The maximum amount of xylitol that wasobserved with this strain was <0.5 g/L. In contrast, no xylitol wasformed when ZW1 (pZB188/Kan) was grown in the same concentration ofglucose but xylose was omitted, and this was true for the other threestrains as well. Consequently, only the experiments that were performedin the presence of both glucose and xylose are shown in FIG. 14.Remarkably, expression of E. coli xylose isomerase in ZW1 greatlyincreased the amount of xylitol that appeared in the fermentation broth,and by 120 hours ZW1 (pZB188/Kan-XylA) had generated five times more ofthis compound than ZW1(pZB188/Kan) (FIG. 14B). As anticipated,expression of xylose isomerase in ZW1 also resulted in the production ofxylulose, since xylose isomerase catalyzes the isomerization of xyloseto xylulose. Note that in this experiment approximately 16% of the totalxylose that was added to the growth medium was converted to xylulose orxylitol. Also note that there is an apparent precursor/productrelationship between these two compounds (xylulose decreased as xylitolincreased), consistent with the hypothesis that GFOR is able to convertxylulose to xylitol under physiological conditions when glucose andxylose are both present in the growth medium.

Similar to ZW1, very little xylitol was generated by ZW1-ΔGFOR in theabsence of the xylose isomerase expression vector (FIG. 14C). The smallamount of xylitol that was formed under these conditions may come froman NADPH-dependent aldose reductase, as suggested by Feldmann et al.(1992 supra). Strikingly, when xylose isomerase was expressed in the ZW1GFOR knockout mutant, no additional xylitol was generated (FIG. 14D), incontrast to the results that were obtained with ZW1 (pZB188/Kan-XylA).Instead, ZW1-ΔGFOR (pZB188/Kan-XylA) produced massive amounts ofxylulose, and the amount of this compound that was formed was verysimilar to the total amount of xylulose and xylitol that was generatedby the corresponding ZW1 stain (i.e. ZW1(pZB188/Kan-XylA)). Theseexperiments clearly demonstrate that GFOR can substantially contributeto xylitol formation in vivo when the enzyme has access to xylulose,which is certainly the case for xylose-utilizing, recombinant strains ofZ. mobilis that are grown in mixtures of glucose and xylose. Theseresults further indicate that NADPH-dependent aldose reductases play aminor role in xylitol production when recombinant strains of Z. mobilisare grown in xylose-containing media, contrary to expectations from theliterature (Feldmann et al, supra; Kim et al, supra).

Example 7 Generation of the ZW658 GFOR Knockout Mutant and Demonstrationthat this Strain does not Produce a Functional GFOR Enzyme

The gene encoding GFOR, which can contribute to xylitol formation in Z.mobilis under physiological conditions when glucose and xylulose areboth available as shown in Example 6, was insertionally-inactivated inZW658 using the suicide construct, pGFORSp-9WW (described in Example 3).All steps in this procedure were identical to those described for theZW1 GFOR knockout mutant in Example 5, including confirmation of thedouble-crossover event with the three sets of PCR primers. The ZW658knockout mutant that was chosen for subsequent experiment describedbelow was named ZW800.

To demonstrate that ZW800 does not produce an enzyme that can generatesorbitol from glucose and fructose, which is the physiological reactionthat is catalyzed by GFOR, the following experiment was performed. Oneand a half milliliter cultures of ZW800 and the parent strain ZW658 weregrown to early stationary phase in 10-ml capped test tubes at 30° C. inliquid medium that contained 75 g/L glucose, 25 g/L xylose, 10 g/L yeastextract, 2 g/L of KH₂PO₄, and 1 g/L MgSO₄. When the cultures reached anOD₆₀₀ of ˜5.5, cells were harvested by centrifugation and thesupernatant was carefully removed and discarded. Next, the cell pelletswere resuspended in 5 ml of fresh growth medium that had the followingcomposition: 110 g/L glucose, 110 g/L fructose, 10 g/L yeast extract, 2g/L of KH₂PO₄, 1 g/L MgSO₄, and 4 g/L KHCO₃. All steps above wereperformed under sterile conditions and the initial pH of the growthmedium was adjusted to 5.8 with concentrated phosphoric acid before thecells were resuspended. The resulting cultures were then grown at 30° C.with gentle agitation (150 rpm) and at times indicated in Table 2,samples were removed for HPLC analysis of the fermentation broth usingthe same procedure that was described in Example 6. The peaks ofinterest for this experiment were glucose, fructose, sorbitol andethanol, and authentic standards of known amount were used to calculatetheir concentrations in the fermentation broth after cells were removedby centrifugation; all concentrations are expressed in g/L in Table 2.

TABLE 2 Sorbitol production in ZW658 and ZW800 - in vivo measurements.Strain Hour Glucose Fructose Sorbitol Ethanol ZW658 0 110 110 0 0 ZW65823 5.99 61.43 45.99 49.36 ZW658 47 1.55 21.98 45.89 69.04 ZW800 0 110110 0 0 ZW800 23 0 60.44 0 73.85 ZW800 47 0 6.79 10.21 96.21

As shown in Table 2, the ZW658 culture consumed almost all of theglucose and about half of the fructose after 23 hr and generatedcomparable amounts of sorbitol and ethanol as major products; the valuesfor the two latter compounds during the first time point were 45.99 g/Land 49.36 g/L, respectively. Thus, more than 40% of the originalfructose was converted to sorbitol by GFOR in the ZW658 culture. Instriking contrast, no sorbitol was detected in the fermentation brothfrom the ZW800 culture after a 23-hr incubation period, and instead theglucose and fructose were almost quantitatively converted to ethanol,which was very close to the theoretical value of 0.51 grams of ethanolper gram of sugar consumed. Note that there was no further increase inthe amount of sorbitol in the ZW658 culture after another 24 hours ofgrowth. This was expected since nearly all of the glucose was depletedearlier and there was no electron donor for the GFOR reaction withfructose. Interestingly, a small amount of sorbitol (10.21 g/L) wasfound in the fermentation broth of the ZW800 culture at the 47-hr timepoint, and this may have been generated by an NADPH-dependent aldosereductase (Feldmann et al., supra) or some other enzyme that remains tobe elucidated. Nevertheless, the above results provide evidence that theSpec^(r)-cassette that was inserted in the middle of the GFOR openreading frame of ZW800 largely, if not entirely, abolished GFOR enzymeactivity.

Further support for this conclusion comes from in vitro experiments withcell-free extracts that were prepared from ZW1, ZW658 and ZW800. Thegoal was to determine if ZW658 can convert xylose to xylitol in theabsence of other added substrates or co-factors, and to see if ZW800 haslost the ability to carry out this reaction as a result of GFORinactivation. There are three requirements for GFOR-mediated xylitolproduction with Z. mobilis cell-free extracts: 1) a sugar electron donorlike glucose or xylose that is able to reduce the GFOR's tightly boundco-factor, 2) xylulose as an electron-acceptor, since it is the compoundthat the enzyme actually reduces to xylitol, and 3) a functional GFORenzyme. If xylulose is not added to the reaction mixture, the cell-freeextract must also contain xylose isomerase to convert xylose toxylulose.

Cell-free extracts were prepared from 100-ml cultures that were grown at33° C. in 250-ml shake flasks that contained 10 g/L yeast extract, 2 g/LKH₂PO₄ and 50 g/L glucose. Cells were harvested by centrifugation at anOD₆₀₀ between 2-3 and were washed twice with ice-cold 50 mM Tris-HCl (pH7.0), 1.0 mM MgCl₂, 1 mM dithiothreitol. The final pellets wereresuspended in 1.0 ml of the same buffer and cells were disrupted bysonication. After cell debris was removed by centrifugation at 4° C.(16,000×g, 60 min), the cell-free extracts were immediately assayed forxylitol production as described below. The 500-μl reactions wereconducted in polypropylene microfuge tubes and contained finalconcentrations of the following components: 50 mM Tris-HCl (pH 7.0), 1.0mM MgCl₂, 1 mM dithiothreitol, 66 mM xylose, and 0.32-0.35 mg ofcell-free extract protein; protein concentrations were determined by theBCA Protein Assay (Pierce) using bovine serum albumen as a standard.Following a 15-hr incubation period at 40° C., reactions were terminatedwith a final concentration of 30 mM pivalic acid and aliquots wereanalyzed by HPLC using a SH1011 column (Showdex) with 0.01N sulfuricacid as the mobile phase. The column temperature was maintained at 50°C. and the flow rate was 1.0 ml/min. The control for this experiment didnot receive cell-free extract, but was otherwise treated identically.

As shown in Table 3, when the ZW1 cell-free extract was added to thereaction mixture, xylose was not converted to xylulose or xylitol duringthe 15-hr incubation period. As already noted, this result was expectedsince ZW1 is a wild type strain that does not express E. coli xyloseisomerase, in contrast to ZW658 and ZW800. In contrast, significantamounts of xylulose and xylitol were generated when the ZW658 cell-freeextract was used, since it contained both enzymes that are necessary forthe formation of these two compounds. Note that in this case nearly 8%of the original 66 mM xylose was used for xylitol production, since twomolecules of xylose are consumed for each molecule of xylitol that isgenerated when xylose is the only GFOR substrate that is added to thereaction mixture. Finally, and most important, the ZW800 cell-freeextract was only able to convert xylose to xylulose, since although itcontained xylose isomerase activity, it lacked GFOR enzyme activity.These results provide additional evidence that ZW800 does not produce afunctional GFOR enzyme and further demonstrate that this protein is ableto use xylose as an electron donor to reduce xylulose to xylitol, aspreviously shown with wild type cell-free extracts that were spiked withpurified xylose isomerase (Danielson supra).

TABLE 3 GFOR-mediated production of xylitol from xylose also requiresxylulose - in vitro measurements Cell-free extract Xylulose (mM) Xylitol(mM) none 0 0 ZW1 0 0 ZW658 9.45 2.6 ZW800 10.63 0

Example 8 Sorbitol is Needed for Growth of ZW800 in ConcentratedMixtures of Glucose and Xylose

The fermentation performance for production of ethanol by ZW800 in aconcentrated mixture of glucose and xylose was tested. The experimentswere conducted in pH-controlled fermentors using a fixed glucose toxylose ratio of ˜5:4 at 97 g/L or 188 g/L of total sugar.

Seed cultures of ZW658 and ZW800 were grown in shake flasks at 37° C. inliquid medium that contained 75 g/L glucose, 25 g/L xylose, 10 g/L yeastextract, 10 g/L KH₂PO₄, 2 g/L (NH₄)₂SO₄, and 1 g/L MgSO₄; initial pH wasadjusted to 5.5 with 4 N KOH. When the OD₆₀₀ reached ˜5.0, 50-mlaliquots of the seed cultures were used to inoculate 1-liter fermentors(BIOSTAT® B-DCU system, Sartorius BBI System Inc., Bethlehem, Pa., USA)that contained 450 ml of growth medium. The final 500-ml culturescontained 5 g/L yeast extract, 2 g/L KH₂PO₄, 2 g/L (NH₄)₂SO₄,1 g/L MgSO₄and either a low concentration of sugar (54 g/L glucose, 43 g/L xylose)or a high concentration of sugar (104 g/L glucose, 84 g/L xylose).Growth was at 33° C. and pH was maintained at 5.5 by automated additionof 4 N KOH. The mixing speed was set at 150 rpm. At various times,aliquots were removed for HPLC analysis of the fermentation broth usingthe same procedure and conditions that were described in Example 6. Thecompounds of interest for this experiment were glucose, xylose, andethanol, and authentic standards of known concentration were used toquantify peaks on the chromatograms. Cell growth was also monitored byfollowing changes in turbidity with a spectrophotometer that was set atan optical density of 600 nm, and the resulting OD₆₀₀ values wereplotted.

As shown in FIG. 15, GFOR inactivation had no effect on growth, sugarconsumption, or ethanol titer when the fermentor contained a lowconcentration of sugar (54 g/L glucose, 43 g/L xylose). However, a bigdifference in fermentation performance was observed when the total sugarconcentration was increased about 2-fold as seen in FIG. 16. ZW658experienced a lag period of about 30 hours, which is typical forxylose-utilizing recombinant strains of Z. mobilis when they are shiftedfrom a dilute mixture of glucose and xylose to a concentrated mixture ofthe same sugars that exceeds ˜180 g/L of total sugar. Following the lagperiod, the cells started to grow and consumed all of the glucose in themedium and about 75% of the xylose, thus resulting in a final ethanoltiter of ˜73 g/L (FIG. 16A). In contrast, the ZW800 culture did notrecover from the lag period even after a 130-hr incubation period (FIG.16B), and this result was obtained on two separate occasions.

Since ZW800 grew well in the dilute mixture of glucose and xylose asshown in FIG. 15, it seemed possible that the inability of this strainto recover in the high sugar mixture was somehow related to osmoticstress. Indeed, GFOR plays a critical role in maintaining osmoticbalance by generating sorbitol when wild type Z. mobilis is transferredto concentrated mixtures of glucose and fructose or high concentrationsof sucrose, which also gives rise to glucose and fructose through theaction of invertase (Loos et al., supra). The sorbitol that is producedby GFOR in the periplasmic space is transported into cells against aconcentration gradient where it accumulates to high levels since it isnot further metabolized. This eliminates the osmotic pressure differenceacross the plasma membrane and restores osmotic balance (Wiegert et al.,supra). However, a prerequisite for GFOR-mediated sorbitol production isthe simultaneous presence of glucose and fructose, and this reactionshould not occur in growth media that lacks fructose. Nevertheless,since sorbitol is the physiologically important product of GFOR and thisenzyme is inactive in ZW800, the effect of adding sorbitol to theconcentrated mixture of glucose and xylose was tested in the experimentdescribed below.

After ˜70 hours in the high sugar mixture (time point designated byvertical arrow in FIG. 16), five 4.5-ml aliquots of the stalled ZW800culture were removed from the fermentor and transferred to 15-ml cappedtest tubes. Four of the tubes were then supplemented with 0-20 mMsorbitol (final concentration), and the total volume of the cultures wasadjusted to 5.0 ml with deionized water in all cases; the sorbitol stocksolution that was used for this experiment was also made up in water. Tocontrol for the 10% dilution of the growth medium when the water andsorbitol were added, nothing was added to the fifth culture. All of thecultures were then incubated at 33° C. with gentle agitation (200 rpm)and growth was monitored spectrophotometrically. The cells started togrow almost immediately when sorbitol was added to the growth medium asshown in FIG. 17, even with the lowest concentration tested (5 mM). Somestimulation of growth was also observed when sorbitol was not added butthe culture was diluted 10% with water, which reduced the total sugarconcentration from 188 g/L to 169 g/L. However, the stimulatory effectof sorbitol on growth was much greater than the effect of dilution.

The rescue of ZW800 growth by sorbitol was completely unexpected sinceZW658 recovered from the lag period and grew well in the concentratedmixture of glucose and xylose, without a known source of fructose. Sincethe latter compound is an obligatory electron acceptor for GFOR-mediatedsorbitol production, it was not apparent that GFOR could synthesizesorbitol or play a role in osmotic balance in media that contain highconcentrations of glucose and xylose. Thus there was no indication thatsorbitol might be an important factor for growth of ZW658 or ZW800 inconcentrated mixtures of glucose and xylose.

Example 9 GFOR Inactivation Improves Ethanol Production from XyloseUnder Process Relevant Conditions

Fermentation performances of ZW658 and ZW800 in concentrated mixtures ofglucose and xylose under process relevant conditions were compared in aside-by-side manner to determine whether GFOR inactivation is abeneficial or detrimental metabolic engineering strategy. Since highconcentrations of glucose and xylose were used in these experiments,sorbitol was added to the medium to allow growth of ZW800. Inexperiments that are not described here, it was also discovered thatsorbitol eliminates the lag period for ZW658. Thus, sorbitolsupplementation of the growth medium provides an ideal way to comparethese two strains under process relevant conditions.

ZW658 and ZW800 were compared under six different conditions using twoconcentrations of total sugars, in the presence and absence of acetate,and for the more concentrated sugar mixture, two different bufferingcapacities were examined. These experiments were conducted with 20-mlcultures that were grown at 30° C. in 50-ml test tubes with gentleagitation (150 rpm). pH was not controlled, but the initial pH of thegrowth medium was adjusted to 5.8 with concentrated phosphoric acidprior to inoculation with the seed culture. The basic growth mediumcontained 10 g/L yeast extract, 2 g/L KH₂PO₄, 1 g/L MgSO₄, 5 mM sorbitoland either 4 g/L (FIGS. 18 and 19) or 8 g/L (FIG. 20) of KHCO₃. Allvalues given above and below are final concentrations after the seedculture was added. The KHCO₃ was used to increase the buffering capacityof the growth medium to minimize the drop in pH that normally occursduring bacterial growth. The carbon source for all of these experimentswas a mixture of glucose and xylose that approximated the ratio of thesetwo sugars in pre-treated corn stover hydrolysate at two differentconcentrations of total sugar. Initial concentrations of glucose andxylose were either 92 g/L and 82 g/L, respectively (FIG. 18) or 107 g/Land 96 g/L (FIGS. 19 and 20). Where indicated, 6 g/L of acetate (aninhibitor that is present in pre-treated corn stover hydrolysate) wasalso present. The seed culture was grown at 30° C. to an OD₆₀₀ of ˜5.0in liquid media that contained 75 g/L glucose, 25 g/L xylose, 10 g/Lyeast extract, 2 g/L KH₂PO₄, 1 g/L MgSO₄, and 1/10^(th) volume was usedto inoculate the experimental cultures. At various times, aliquots wereremoved for HPLC analysis of the fermentation broth as previouslydescribed in Example 6. The compounds of interest for this experimentwere glucose, xylose, ethanol and xylitol, and all values are reportedin g/L. Since virtually all of the glucose was consumed before the firsttime points were taken, the values for this sugar were not plotted inthe graphs.

From the experiments that are shown in FIGS. 18-20, it is clear thatZW800 outperformed ZW658 under all conditions tested as judged by twodifferent criteria: (a) the total amount of xylose that was consumedduring the course of the experiment, and (b) the maximum ethanol titerthat was achieved. It is also evident from this data that the beneficialeffects of GFOR inactivation largely occurred during the late stage offermentation when the most stressful conditions were encountered (i.e.after all of the glucose was depleted and the ethanol concentrationstarted approaching toxic levels). Indeed, the most striking differencesbetween the two strains were observed when an inhibitory concentrationof acetate was present in the growth medium, which constitutes anadditional stress. The average increase in ethanol titer for ZW800 inthe presence of acetate for the three sets of experimental conditionswas 10.2% with values ranging from 4.4% to 13.7%. ZW800 also producedmore ethanol than ZW658 in the absence of acetate in all threeexperiments, with the average increase in this case being 3.2%. Asanticipated, ZW658 converted significant amounts of xylose to theunwanted by-product xylitol, and the highest levels of this compoundwere observed when conditions were the most stressful (i.e. during thelate stages of fermentation and when acetate was present in the growthmedium). For example, in the experiments with acetate that are shown inFIGS. 18-20, ZW658 converted 8.1%, 8.3% and 9.9% of the total xylosethat was consumed to xylitol. In contrast, no xylitol was found in theZW800 cultures under any of the conditions that were tested. Theseresults clearly show that GFOR inactivation is beneficial to xylosemetabolism for ethanol production under process relevant conditions,especially in the presence of inhibitory concentrations of acetate. Thetest tube experiments that are shown in FIGS. 18 and 19 were performedtwice and virtually identical results were obtained.

Another side-by-side experiment with ZW800 and ZW658 in a concentratedmixture of glucose and xylose with acetate was performed usingpH-controlled fermentors. By-products of metabolism such as organicacids and carbon dioxide produced by Z. mobilis can lower the pH, of thegrowth medium which increases the ratio of acetic acid to acetate, andit is known that the protonated species is the compound that actuallyinhibits bacterial growth (Kim et al, (2000) Applied Biochemistry andBiotechnology, 84-86:357-370). Thus, pH control is very important inlarge-scale fermentation, since a drop in pH from 5.8 to 5.0 wouldresult in about a 5-fold increase in the concentration of acetic acid.

Seed cultures for ZW800 and ZW658 were grown in shake flasks at 37° C.in liquid medium that contained 75 g/L glucose, 25 g/L xylose, 10 g/Lyeast extract, 10 g/L KH₂PO₄, 2 g/L (NH₄)₂SO₄, and 1 g/L MgSO₄; initialpH was adjusted to 5.5 with 4 N KOH. When the OD₆₀₀ reached ˜5.0, 50-mlaliquots of the seed cultures were used to inoculate 1-liter fermentors(BIOSTAT® B-DCU system, Sartorius BBI System Inc., Bethlehem, Pa., USA)that contained 450 ml of growth medium. The final 500-ml culturescontained 92 g/L glucose, 97 g/L xylose, 10 g/L yeast extract, 2 g/LKH₂PO₄, 10 mM sorbitol and 7.2 g/L of acetate. Growth was at 33° C. andpH was maintained at 5.5 by automated addition of 4 N KOH; the mixingspeed was 150 rpm. At various times, aliquots were removed for HPLCanalysis of the fermentation broth using the same procedure that isdescribed in Example 6. The compounds of interest for this experimentwere glucose, xylose, ethanol and xylitol, and cell growth (OD₆₀₀) wasalso monitored. FIG. 21 shows the full time course for these parametersfor the fermentor run with the ZW800 culture and Table 4 summarizesend-point values for xylose, ethanol and xylitol for both strains.

TABLE 4 End-point values for xylose, ethanol, and xylitol inpH-controlled fermentors with ZW800 and ZW658. ZW658 ZW800 Ethanol (g/L)65.95 72.31 Xylose consumed (g/L) 60.71 69.14 Xylitol (g/L) 3.92 0Ethanol yield (g ethanol/g sugar) 0.43 0.45

Similar to the test tube results with acetate (FIGS. 18-20), ZW800consumed 14% more xylose and generated 9.6% more ethanol than ZW658 inthe pH-controlled fermentors (FIG. 21). The ethanol yield for ZW800 wasalso ˜5% higher since this strain did not produce any detectablexylitol. In contrast, the final concentration of xylitol in fermentationbroth for ZW658 was 3.92 g/L, which represents ˜6.5% of the total xylosethat was consumed during the course of the experiment. These experimentsprovide additional evidence that GFOR inactivation improves ethanolproduction from xylose by eliminating xylitol formation. As alreadynoted, the unwanted by-product xylitol interferes with xylose metabolismin at least two different ways and inhibits bacterial growth, whichresults in lower levels of ATP. Thus, when GFOR generates xylitol itreduces the ability of Z. mobilis to cope with all of the otherenergy-consuming stresses that it normally encounters during ethanolproduction from lignocellulose feedstocks. Since ZW800 does not have tocontend with xylitol-related stresses in contrast to ZW658, it consumesmore xylose, produces more ATP and generates more ethanol during thelate stage of fermentation when the highest level of stress isencountered.

Example 10 Removing the Selectable Marker from ZW800 andCharacterization of the Resulting Strain, ZW801-4

Generation of the Cre-Expression Construct, pZB188-Kan/Cre

As described in Example 3, the Spec^(r)-cassette that was inserted intothe GFOR open reading frame in ZW800 is sandwiched between two wild typeloxP sites. This arrangement makes it easy to remove the selectablemarker from the chromosome by using Cre Recombinase (Sternberg andHamilton (1981) J. Mol. Biol. 150:467-486; Lee and Saito, supra; Trinhet al. (2000) Journal of Immunological Methods 244(2):185-193). In orderto do this, however, it was first necessary to generate a Cre Expressionvector that can replicate in Z. mobilis (FIG. 22). The precursor for theCre Expression vector was pZB188/Kan, which was described in detail inExample 4. Briefly, pZB188/Kan is a shuttle vector that can replicate inE. coli and Z. mobilis because it has an origin of replication for bothbacterial species. It also contains a DNA fragment that confersresistance to kanamycin (i.e. a Kan^(r)-cassette). pZB188/Kan wasdouble-digested with NcoI and NotI, and the large vector fragment waspurified by agarose gel electrophoresis. The next step was to generate aCre-expression cassette and this was accomplished by PCR using primers17 and 18. The DNA template that was used for amplification of theCre-expression cassette was a plasmid that contained the full-lengthgene for the bacteriophage PI Cre Recombinase including its promoter(Sternberg et al. (1986) J. Mol. Biol. 187(2):197-212)).

Primer 17 (SEQ ID NO:35) CTACTCATccatggCATCTTGAGCTTGAGAAAAACC Primer 18(SEQ ID NO:36) CATCTTACTgcggccgcTTAATGGCTAATCGCCATCTTC

The underlined bases of Primer 17 (forward primer) hybridize to nt286-307 of the GenBank accession number X03453 sequence which is ˜200 bpupstream from the Cre start codon, while the lower case letterscorrespond to an NcoI site that was added to the 5′ end of the primer.The underlined bases of Primer 18 (reverse primer) bind at the other endof the Cre open reading frame to nt 1523-1503 of the GenBank accessionnumber X03453 sequence, while the lower case letters indicate a NotIsite that was added to the 5′ end of this primer. The 1238 bp PCRproduct was double-digested with NcoI and NotI, and the resulting DNAfragment, which contains the complete open reading frame for CreRecombinase and its putative promoters (Sternberg et al., 1986 supra),was purified by agarose gel electrophoresis. The Cre-expression cassettewas then inserted between the NcoI and NotI sites of the pZB188/Kan DNAfragment that was described above in a standard ligation reaction. Analiquot of the ligation reaction mixture was electroporated into E. coliDH10B, and the transformed cells were plated on LB media that containedkanamycin (50 μg/ml); growth was at 37° C. Plasmid DNA was isolated fromone of the kanamycin-resistant transformants, and this preparation wasthen introduced into E. coli JM110 (dcm⁻¹, dam⁻) to obtainnon-methylated plasmid DNA for subsequent transformation of Z. mobilis(see below). A plasmid map of the Cre Expression vector pZB188/Kan-Creis shown in FIG. 22.

Cre Treatment to Remove the Selectable Marker from the Chromosome ofZW800 and Curing of the Cre Expression Vector

The Cre Recombinase of bacteriophage P1 (Cre) is able to recognize aspecific 34-bp DNA sequence, a “loxP site”, which contains two 13-bpinverted repeats that flank an 8-bp asymmetric core (Sternberg andHamilton, 1981 supra; Lee and Saito, supra; Trinh et al. supra). Cre isalso able to excise any intervening DNA fragment that is situatedbetween two identical loxP sites, and the excision reaction is veryrapid. To remove the Spec^(r)-cassette from the GFOR open reading frame,the Cre Expression vector (pZB188/Kan-Cre) was introduced into ZW800.The transformation protocol was essentially as described in Example 5,but after the recovery period the cells were plated on MMG media thatcontained 350 mg/ml of kanamycin, which is the selective agent for theCre Expression vector. The primary transformants that were recoveredfrom this process were no longer resistant to spectinomycin, since theSpec^(r)-cassette that was removed from the chromosome by Cre is acircular piece of DNA that cannot replicate in Z. mobilis. After a 48-hrincubation period at 30° C. under anaerobic conditions, two of theKan^(r)/Spec^(s) primary transformants were subjected to the Creplasmid-curing process. Although pZB188/Kan-Cre can replicate in Z.mobilis it is relatively easy to cure this plasmid by growing the cellsin media that does not contain kanamycin. To cure the Cre Expressionvector in the present invention, the cells were grown at an elevatedtemperature (37° C.) in liquid MMG media that did not contain kanamycin;the cells were transferred to fresh growth media with the samecomposition every 24-36 hours. After at least 50 generations hadoccurred, single colonies were isolated on MMG plates, and five coloniesfrom both of the original primary transformants were randomly selectedfor further characterization. As anticipated, none of these colonieswere able to grow on MMG plates that contained kanamycin (350 μg/ml) orspectinomycin (200 μg/ml). Although the inability to grow on kanamycinwas a good indication that the plasmid-curing process was successful,this conclusion was confirmed by colony PCR using primers that hybridizeto the Cre-expression cassette. Based on these experiments, three of theCre-treated, plasmid-cured ZW800 derivatives were selected for furthercharacterization and these strains are referred to below as ZW801-4,ZW801-5 and ZW801-6.

To see how well these strains perform in a concentrated mixture ofglucose and xylose in the presence of an inhibitory concentration ofacetate, shake flask experiments were performed. ZW658 and ZW800 werealso included in this analysis. The seed cultures were grown at 30° C.to an OD₆₀₀ of ˜3.0 in liquid media that contained 75 g/L glucose, 25g/L xylose, 10 g/L yeast extract, 2 g/L KH₂PO₄, 1 g/L MgSO₄, and a 10%inoculum was used for the 15-ml experimental cultures. The latter weregrown in 50-ml test tubes at 30° C. with gentle agitation (150 rpm). Thegrowth media contained 10 g/L yeast extract, 2 g/L KH₂PO₄, 1 g/L MgSO₄,5 mM sorbitol, 40 mM KHCO₃, 95 g/L glucose, 90 g/L xylose, and 7.7 g/Lacetate; the initial pH was adjusted to 5.8 with concentrated phosphoricacid. At various times, aliquots of the cultures were removed for HPLCanalysis of the fermentation broth as previously described in Example 6.The compounds of interest for this experiment were glucose, xylose,ethanol and xylitol, and all values are reported in g/L.

As shown in Table 5, ZW658 produced 66.35 g/L of ethanol and left behind40.6 g/L of residual xylose. ZW658 also produced 3.19 g/L of theunwanted by-product xylitol since it has a functional GFOR enzyme.Similar to what was previously observed in other side-by-sideexperiments, ZW800 consumed 17% more xylose and produced 6.2% moreethanol than ZW658, and it did not produce any detectable xylitol.Although slightly better results were obtained with ZW801-4 and ZW801-6,these differences are probably within experimental error and are notstatistically significant. The relatively poor performance of ZW801-5that was observed in this experiment is not understood and was notfurther investigated. Based on these results, strain ZW801-4 wasselected for further analysis.

TABLE 5 Shake Flask Experiments with ZW658, ZW800, ZW801-4, ZW801-4, andZW801-5 in High Sugar Plus Acetate Strain Hours Glucose Xylose XylitolEthanol ZW658 0 95.7 89.3 0 3.2 ZW658 15.5 27.75 80.93 0 37.39 ZW658 380 42.71 1.85 66.53 ZW658 62 0 40.6 3.19 66.35 ZW800 0 95.7 89.3 0 3.2ZW800 15.5 30.64 81.36 0 36.05 ZW800 38 0 37.29 0 69.82 ZW800 62 0 32.340 70.47 ZW801-4 0 95.7 89.3 0 3.2 ZW801-4 15.5 28.04 80.82 0 37.75ZW801-4 38 0 36.13 0 70.54 ZW801-4 62 0 30.28 0 71.25 ZW801-5 0 95.789.3 0 3.2 ZW801-5 15.5 55.61 85.62 0 21.86 ZW801-5 38 0 46.83 0 64.92ZW801-5 62 0 39.54 0 66.19 ZW801-6 0 95.7 89.3 0 3.2 ZW801-6 15.5 32.3482.02 0 34.89 ZW801-6 38 0 36.39 0 70.64 ZW801-6 62 0 29.55 0 71.74

To confirm the results from the shake flask experiments that suggestedthat ZW801-4 performed at least as well as ZW800, these two strains werecompared under pH-controlled conditions. The seed cultures were grown at30° C. in media that contained 75 g/L glucose, 25 g/L xylose, 10 g/Lyeast extract, 2 g/L KH₂PO₄, 1 g/L MgSO₄. When the OD₆₀₀ reached ˜4.6,17-ml aliquots of the seed cultures were used to inoculate thepH-controlled bioreactors that contained 153 ml of growth medium. Thefinal 170 ml cultures contained 105 g/L glucose, 100 g/L xylose, 10 g/Lyeast extract, 2 g/L KH₂PO₄, 1 g/L MgSO₄, 5 mM sorbitol and 7.2 g/L ofacetate. Growth was at 33° C. and pH was maintained at 5.5 by automatedaddition of 4 N KOH; the mixing speed was ˜150 rpm. At various times,aliquots of the cultures were removed from the bioreactors for HPLCanalysis of the fermentation broth as described above, and OD₆₀₀ wasalso monitored. Under these experimental conditions, the growth curvesfor ZW800 and ZW801-4 were almost superimposable (FIG. 23A). The timecourses for glucose and xylose consumption were also virtuallyidentical, and both strains produced the same amount of ethanol withsimilar kinetics (FIG. 23B). Furthermore, neither of these strainsproduced any detectable xylitol. Based on these observations, weconclude that removing the Spec^(r)-cassette from the GFOR open readingframe did not restore or partially restore GFOR enzyme activity, andthat this manipulation did not adversely effect fermentationperformance. Although ZW800 and ZW801-4 both performed better than theparent strain (ZW658), which has a functional GFOR enzyme, the preferredstrain for commercial applications is ZW801-4 since it does not containa foreign gene that confers resistance to an antibiotic.

Sequence analysis of genomic DNA from ZW801-4 provided unequivocal proofthat the correct Cre excision event had indeed occurred. The completenucleotide sequence of the disrupted GFOR open reading in ZW801-4 (fromthe original start codon through the original stop codon) is given inSEQ ID NO:37, and FIG. 24 shows an alignment of the translated mutantsequence with the wild type GFOR protein; the latter is coded for by thereverse complement of nt 683751-685052 of GenBank accession numberAE008692. As anticipated, Cre excision of the Spec^(r)-cassette left asingle wild type loxP site in the middle of the GFOR open reading frame,and this insertion event resulted in an in-frame stop codon thatprematurely truncates the protein; the location of the “lox scar” isindicated by the gray highlighted residues. The mutant nucleotidesequence is also missing ˜72 bp of the original wild type GFORnucleotide sequence in the same location as a result of the design ofthe suicide construct.

1. A process for producing ethanol comprising, a) providing arecombinant Zymomonas strain capable of utilizing xylose to produceethanol, said strain comprising at least one genetic modification whichreduces glucose-fructose oxidoreductase activity, and b) culturing thestrain of (a) in a medium comprising xylose whereby xylose is convertedto ethanol.
 2. The process according to claim 1, wherein the strain of(a) improves conversion of xylose to ethanol relative to a recombinantZymomonas strain without at least one genetic modification which reducesglucose-fructose oxidoreductase activity.
 3. The process of claim 2,wherein the strain of (a) improves one or more of rate, titer or yield,of xylose conversion to ethanol relative to a recombinant Zymomonasstrain without at least one genetic modification which reducesglucose-fructose oxidoreductase activity.
 4. The process according toclaim 1, wherein the strain of (a) produces a reduced amount of xylitolwhen metabolizing xylose to ethanol as compared to a Zymomonas strainwithout at least one genetic modification which reduces glucose-fructoseoxidoreductase activity.
 5. The process according to claim 4, whereinthe strain of (a) produces substantially no xylitol when metabolizingxylose to ethanol.
 6. The process according to claim 1, wherein therecombinant Zymomonas strain of (a) is selected from the groupconsisting of ZW800. ZW801-4 and ZW801-6.
 7. The process of claim 1,wherein the medium comprises a mixture of sugars including xylose and asugar alcohol selected from the group consisting of sorbitol, mannitol,galactitol, ribitol, and a mixture thereof, wherein the mixture ofsugars is at a concentration of at least about 120 g/L and wherein thesugar alcohol is at a final concentration that is between about 2 mM andabout 100 mM.
 8. The process of claim 7, wherein the sugar alcohol is ina concentration that is between about 5 mM and about 20 mM.